Progression elevated Gene-3 and uses thereof

ABSTRACT

This invention provides a vector suitable for introduction into a cell, comprising: a) an inducible PEG-3 regulatory region; and b) a gene encoding a product that causes or may be induced to cause the death or inhibition of cancer cell growth. In addition, this invention further provides the above-described vectors, wherein the inducible PEG-3 regulatory region is a promoter. This invention further provides the above-described vectors, wherein the gene encodes an inducer of apoptosis. In addition, this invention provides the above-described vectors, wherein the gene is a tumor suppressor gene. In addition, this invention provides the above-described vectors, wherein the gene encodes a viral replication protein. This invention also provides the above-described vectors, wherein the gene encodes a product toxic to cells or an intermediate to a product toxic to cells. In addition, this invention provides the above-described vectors, wherein the gene encodes a product causing enhanced immune recognition of the cell. This invention further provides the above-described vectors, wherein the gene encodes a product causing the cell to express a specific antigen.

[0001] The invention disclosed disclosed herein was made with UnitedStates Government support under National Institute of Health Grant CA35675. Accordingly, the United States Government has certain rights inthis invention.

[0002] Throughout this application, various references are referred towithin parentheses. Disclosures of these publications in theirentireties are hereby incorporated by reference into this application tomore fully describe the state of the art to which this inventionpertains. Full bibliographic citations for these references may be foundat the end of this application, preceding the claims.

BACKGROUND OF THE INVENTION

[0003] The carcinogenic process involves a series of sequential changesin the phenotype of a cell resulting in the acquisition of newproperties or a further elaboration of transformation-associated traitsby the evolving tumor cell (1-4). Although extensively studied, theprecise genetic mechanisms underlying tumor cell progression during thedevelopment of most human cancers remain enigmas. Possible factorscontributing to transformation progression, include: activation ofcellular genes that promote the cancer cell phenotype, i.e., oncogenes;activation or modification of genes that regulate genomic stability,i.e., DNA repair genes; loss or inactivation of cellular genes thatfunction as inhibitors of the cancer cell phenotype, i.e. tumorsuppressor genes; and/or combinations of these genetic changes in thesame tumor cell (1-6). A useful model system for defining the geneticand biochemical changes mediating tumor progression is the type 5adenovirus (Ads)/early passage rat embryo (RE) cell culture system(1,7-14). Transformation of secondary RE cells by Ads is often asequential process resulting in the acquisition of and furtherelaboration of specific phenotypes by the transformed cell (7-10).Progression in the Ad5-transformation model is characterized by thedevelopment of enhanced anchorage-independence and tumorigenic potential(as indicated by a reduced latency time for tumor formation in nudemice) by progressed cells (1,10). The progression phenotype inAdS-transformed RE cells can be induced by selection for growth in agaror tumor formation in nude mice (7-10), referred to asspontaneous-progression, by transfection with oncogenes (13), such asHa-ras, v-src, v-raf or the E6/E7 region of human papillomavirus type(HPV)-18, referred to as oncogene-mediated progression, or bytransfection with specific signal transducing genes (14), such asprotein kinase C, referred to as growth factor-related, gene-inducedprogression.

[0004] Progression, induced spontaneously or after gene transfer, is astable cellular trait that remains undiminished in Ad5-transformed REcells even after extensive passage (>100) in monolayer culture (13).However, a single-treatment with the demethylating agent 5-azacytidine(AZA) results in a stable reversion in transformation progressionin >95% of cellular clones (10,13,14). The progression phenotype is alsosuppressed in somatic cell hybrids formed between normal or unprogressedtransformed cells and progressed cells (11-13). These findings suggestthat progression may result from the activation of specificprogression-promoting genes or the selective inhibition ofprogression-suppressing genes, or possibly a combination of bothprocesses.

[0005] The final stage in tumor progression is acquisition bytransformed cells of the ability to invade local tissue, survive in thecirculation and recolonize in a new area of the body, i.e., metastasis(15-17). Transfection of a Ha-ras oncogene into cloned rat embryofibroblast (CREF) cells (18) results in morphological transformation,anchorage-independence and acquisition of tumorigenic and metastaticpotential (19-21). Ha-ras-transformed CREF cells exhibit major changesin the transcription and steady-state levels of genes involved insuppression and induction of oncogenesis (21,22). Simultaneousoverexpression of the Ha-ras suppressor gene Krev-1 inHa-ras-transformed CREF cells results in morphological reversion,suppression of agar growth capacity and a delay in in vivo oncogenesis(21). Reversion of transformation in Ha-ras+Krev-1 transformed CREFcells correlates with a return in the transcriptional and steady-statemRNA profile to that of untransformed CREF cells (21,22). Following longlatency times, Ha-ras+Krev-1 transformed CREF cells form both tumors andmetastases in athymic nude mice (21). The patterns of gene expressionchanges observed during progression, progression suppression and escapefrom progression suppression supports the concept of “transcriptionalswitching” as a major component of Ha-ras-induced transformation(21,22).

[0006] To identify potential progression inducing genes with elevatedexpression in progressed versus unprogressed AdS-transformed cells weused subtraction hybridization (13,23). This approach resulted in thecloning of PEG-3 that is expressed at elevated levels in progressedcells (spontaneous, oncogene-induced and growth factor-related,gene-induced) than in unprogressed cells (parental Ad5-transformed,AZA-suppressed, and suppressed hybrids). Transfection of PEG-3 intounprogressed parental Ad5-transformed cells induces the progressionphenotype, without significantly altering colony formation in monolayerculture or affecting cell growth. PEG-3 expression is also elevatedfollowing DNA damage and oncogenic transformation of CREF cells byvarious oncogenes. Sequence analysis indicates that PEG-3 has 73 and 68%nucleotide (nt) and 59 and 72% amino acid (aa) similarities,respectively, with the gadd34 and MyD116 gene. However, unlike gadd34and MyD116 that encode proteins of ˜65 and ˜72 kDa, respectively, PEG-3encodes a protein of ˜50 kDa with only ˜28 and ˜40% aa similarities togadd34 and Myd116, respectively, in its carboxyl terminus. These resultsindicate that PEG-3 represents a new member of the gadd34/MyD116 genefamily with both similar and distinct properties. Unlike gadd34 andMyD116, which dramatically suppress colony formation (24), PEG-3 onlymodestly alters colony formation following transfection, i.e., ≦20%reduction in colony formation in comparison with vector transfectedcells. Moreover, a direct correlation only exists between expression ofPEG-3, and not gadd34 or Myd116, and the progression phenotype intransformed rodent cells. These findings provide evidence for apotential link between constitutive induction of a stress response,characteristic of DNA damage, and induction of cancer progression.

SUMMARY OF THE INVENTION

[0007] This invention further provides an inducible PEG-3 regulatoryregion functionally linked to a gene encoding a product that causes ormay be induced to cause the death or inhibition of cancer cell growth.

[0008] In addition, this invention further provides the above-describedvectors, wherein the inducible PEG-3 regulatory region is a promoter.

[0009] This invention further provides the above-described vectors,wherein the gene encodes an inducer of apoptosis.

[0010] In addition, this invention provides the above-described vectors,wherein the gene is a tumor suppressor gene.

[0011] In addition, this invention provides the above-described vectors,wherein the gene encodes a viral replication protein.

[0012] This invention also further provides the above-described vectors,wherein the gene encodes a product toxic to cells or an intermediate toa product toxic to cells.

[0013] In addition, this invention provides the above-described vectors,wherein the gene encodes a product causing enhanced immune recognitionof the cell.

[0014] This invention further provides the above-described vectors,wherein the gene encodes a product causing the cell to express aspecific antigen.

[0015] In addition, this invention provides a method of treating cancerin a subject, comprising: a) administering one of the above-describedvectors to the subject; and b) administering an antibody or a fragmentof an antibody to the the above-described antigen to the subject.

BRIEF DESCRIPTION OF THE FIGURES

[0016]FIG. 1: Northern blot illustrating PEG-3 expression inAd5-transformed RE cells displaying different stages of transformationprogression. Fifteen μg of cellular RNA isolated from the indicated celltypes, were electrophoresed, transferred to nylon membranes andhybridized with an ˜700 bp 3′ region of the PEG-3 gene (top) and thenstripped and probed with GAPDH (bottom).

[0017]FIG. 2: Northern blot illustrating PEG-3 expression in gammairradiated and oncogene transformed CREF cells. The experimentalprocedure was as described in the legend to FIG. 1. CREF cells weregamma irradiated with 10 Gy and RNA was isolated 4 and 24 hr later.Fifteen μg of cellular RNA isolated from the indicated cell types, wereelectrophoresed, transferred to nylon membranes and hybridized with an˜700 bp 3′ region of the PEG-3 gene (top) and then stripped and probedwith GAPDH (bottom).

[0018] FIGS. 3A and 3B: Predicted amino acid sequences of the rat PEG-3,gadd34 and MyD116 proteins. Sequences shared by the three genes areshaded. PEG-3 encodes a putative protein of 457 aa (MW of ˜50 kDa), thegadd34 gene encodes a putative protein of 589 aa (MW ˜65 kDa) and theMyD116 gene encodes a putative protein of 657 aa (MW of ˜72 kDa).

[0019]FIG. 4: Results of in vitro translation of the rat PEG-3 gene.Lane Luciferase is the in vitro translation of the luciferase gene (˜61kDa), positive control. The blank lane contains the same reactionmixture without mRNA, negative control. Lane PEG-3 contains thetranslated products of this cDNA. Rainbow protein standards (Amersham)were used to determine the sizes of the in vitro translated products.

[0020]FIG. 5: An autoradiogram illustrating the transcription of the ratPEG-3, gadd34 and MyD116 genes as a function of DNA damage andtransformation progression. Nuclear run-on assays were performed todetermine comparative rates of transcription. Nuclei were isolated fromCREF cells treated with MMS (100 μg/ml for 2 hr followed by growth for 4hr in complete medium) or gamma irradiation (10 Gy followed by 2 hrgrowth in complete medium). DNA probes include, PEG-3 (1), MyD116 (2),gadd34 (3), GAPDH (4) and pBR322 (5).

[0021]FIG. 6: Histogram illustrating the effect of transfection withPEG-3, mda-7 and p21 (mda-6) on colony formation of E11 and E11-NMTcells in monolayer culture. Target cells were transfected with 10 μg ofa Zeocin vector (pZeoSV), the PEG-3 gene cloned in pZeoSV (PEG-3), thepREP4 vector, the mda-7 gene cloned in pREP4 (mda-7) and the mda-6 (p21)gene cloned in pREP4 (p21 (mda-6)), as indicated. Data represents theaverage number of Zeocin or hygromycin (pREP4 transfection) resistantcolonies ±S.D. for 4 plates seeded at 1×10⁵ cells/6-cm plate.

[0022]FIG. 7: Histogram illustrating the effect of stable PEG-3expression on anchorage independent growth of E11 cells. Agar cloningefficiency of E11, Zeocin resistant pZeoV (vector) transfected E11 andZeocin resistant pZeoPEG transfected E11 cells. Average number ofcolonies developing in 4 replicate plates ±S.D.

[0023]FIG. 8: Autoradiogram illustrating the expression of PEG-3, Ad⁵E1A and GAPDH RNA in pZeoPEG transfected E11 cells. The experimentalprocedure was as described in the legend to FIG. 1. Blots were probedsequentially with PEG-3 (top), Ad⁵ E1A (middle) and GAPDH (bottom). TheE11-ZeoPEG clones are the same clones analyzed foranchorage-independence in FIG. 7.

[0024]FIG. 9: Autoradiogram showing PEG-3 expression in normal humanmelanocyte and melanoma cell lines. Fifteen μg of cellular RNA isolatedfrom the indicated cell types, were electrophoresed, transferred tonylon membranes and hybridized with an ˜700 bp 3′ region of the PEG-3gene (top) and then stripped and probed with GAPDH (bottom). Cell typesinclude: FM516-SV, normal human melanocyte immortalized with the SV40T-antigen; MeWo, WM239, C8161, F0-1 and Ho-1, metastatic human melanoma;WM35, early radial growth phase (RGP) primary human melanoma; and WM278,early vertical growth phase (VGP) primary human melanoma.

[0025]FIG. 10: Autoradiogram showing the effect of treatment with DNAdamaging agents on PEG-3 expression in human melanoma cells. Theindicated cell type was exposed to methyl methanesulfonate (MMS) (100μg/ml for 2 hr and then grown in medium) lacking MMS for 2 hr) or gammairradiation (JR) (10 gy and cells were grown for 4 or 24 hr in medium)prior to RNA isolation. Fifteen μg of cellular RNA isolated from theindicated cell types and conditions, were electrophoresed, transferredto nylon membranes and hybridized with an ˜700 bp 3′ region of the PEG-3gene (top) and then stripped and probed with GAPDH (bottom). HO-1 andF0-1 cells express wild-type p53 protein (p53 wt) and SK MEL 110expresses a mutant p53 (p53 mut).

[0026]FIG. 11: Nucleotide sequence of rat Progression Elevated Gene-3(PEG-3). The initiation and termination codons are underlined.

[0027]FIG. 12: Amino acid sequence of Progression Elevated Gene-3(PEG-3). PEG-3 protein contains 457 amino acids and with M.W. ofapproximately 50 kDa.

[0028] FIGS. 13A-13C: Nucleotide and amino acid sequence of a humanPEG-3 cDNA.

[0029]FIG. 14: Sequence of the rat PEG-3 promoter. This region of DNAconsists of 2,616 nucleotides. This DNA sequence contains the putativeinitiation site of transcription of the rat PEG-3 gene. For luciferaseassays a ˜2,200 nucleotide region of the PEG-3 promoter was cloned intoa luciferase reporter vector.

[0030]FIG. 15: Relative PEG-3 promoter luciferase activity inunprogressed (E11 and E11-NMT-AZA) and progressed (E11-NMT and E11-ras)transformed rodent cells.

[0031]FIG. 16: Relative PEG-3 promoter luciferase activity in normal andtransformed CREF cells. These include unprogressed and non-tumorigenic(CREF, Rat 1, CREF-ras+Krev-1 B1, and CREF-wt3A (Ad5) and progressed andoncogenic (CREF-Trans 6:4 NMT, CFEF-Ha-ras, CREF-ras+Krev-1 BIT,CREF-ras+Krev-1 B1M, CREF-A2 (H5hr1), CREF-src, CREF-HPV-18, CREF-rafand CREF-PKC B1) cells. * Indicates non-tumorigenic cell type.

[0032]FIG. 17: Induction of PEG-3 promoter luciferase activity in CREFand CREF-PEG-Luc cells after MMS (100 μg/ml) treatment. CREF (O) andCREF-PEG-Luc (X).

[0033]FIG. 18: The Rapid Promoter Screening (RPS) assay system foridentifying compounds and experimental conditions regulating importantphysiological processes. The systems outlined above represent sensitivebiosensor monitoring approaches for defining conditions and compoundsthat can regulate cellular phenotype, thereby altering the functionalityof the PEG-3 promoter. Briefly, the PEG-3 promoter is linked to areporter gene, such as luciferase, and stable cell clones are generatedthat contain either an inducible PEG-Luc gene (CREF) or a suppressiblePEG-Luc gene (E11-NMT or various transformed CREF cells, including 4NMT,CREF-ras, CREF-src, CREF-HPV etc.). The PEG-3-Luc containing clones canthen be used as sensitive indicators of alterations in cellularphysiology resulting from DNA damage, induction of cancer progression,induction of oncogenic transformation, treatment with chemopreventionagents, inhibitors of cancer progression, inhibitors of angiogenesis andagents specifically involved in regulating defined oncogenic pathways.The RPS approach can be adapted for manual or rapid automated screening.

[0034]FIG. 19: Northern blot analysis of human PEG-3. A 500 bp probefrom the 3′ end of the human PEG-3 gene was used to probe a Northernblot containing the following tumor and normal cell lines.

[0035] MCF-7, T47D: human breast carcinoma

[0036] LNCaP, PC-3: human prostate carcinoma

[0037] T98G, GBM-18: human glioblastoma

[0038] HO-1, LO-1, SH-1, C8161, FO-1: human melanoma

[0039] NhPEC: normal human prostate epithelial cells (Clonetics)

[0040] HBL-100: immortalized normal human breast cells

[0041] HeLa: human ovarian carcinoma

[0042] REF(RAD): irradiated CREF-Trans 6 cells

[0043] E11, E11NMT: Sprague Dawley rat embryo cells transformed withmutant adenovirus H5ts125

[0044] 4NMT: CREF-Trans 6 cells transformed with LNCaP high molecularweight DNA

[0045] HONE-1: human nasopharyngeal carcinoma

[0046]FIG. 20: Northern blot analysis of human PEG-3. A 500 bp probefrom the 3′ end of the human PEG-3 gene was used to probe a normal humantissue blot (Clonetics)

[0047]FIG. 21: Titration of CREF-Trans 6 4NMT cells containing the ratPEG-3 promoter/luciferase reporter gene. Cells were grown in 96 wellplates, lysed as described and the luciferase activity was read in aluminometer.

[0048]FIG. 22: Effects of an antisense oligonucleotide to the PTI-1oncogene on expression of the rat PEG-3 promoter/luciferase reportergene in CREF-Trans 6 4NMT cells. 4NMT cells were treated with anantisense oligonucleotide to the bridge region of the PTI-1 gene for 24,48, and 72 hours. Cells were lysed and luciferase activity wasdetermined using a luminometer.

[0049]FIG. 23: Luciferase activation in the presence of variousoncogenes expressed in CREF-Trans 6 cells transfected with the rat PEG-3promoter/luciferase reporter. CREF-Trans 6 cells were stably transfectedwith the following oncogenes: ras, src, and PTI-1. 4NMT cells weretransfected with high molecular weight DNA from the human prostatecarcinoma cell line LNCaP and expresses PTI-1. Cells grown in 96 wellmicrotiter plates, lysed, and assayed for luciferase activity.

[0050]FIG. 24: Schematic diagram of specific deletion mutants in the ratPEG-3 promoter. The deletion mutants are labeled as 2 to 11, with 1being the unmodified promoter. Numbers in brackets indicate thebeginning nucleotide remaining after deletion from the 5Õ region of therat PEG-3 promoter. The TATA box is located at nucleotide 1751 from the5′ region of the promoter.

[0051]FIG. 25: Relative PEG-3 promoter luciferase activity in E11 cellsafter transfection with an intact rat PEG-3 promoter (1) and variousdeletion mutants of the rat PEG-3 promoter (2 through 11). Furtherinformation in FIG. 24.

[0052]FIG. 26: Relative PEG-3 promoter luciferase activity in E11-NMTcells after transfection with an intact rat PEG-3 promoter (1) orvarious deletion mutants of the rat PEG-3 promoter (2 through 11).Further information in FIG. 24.

[0053]FIG. 27: Comparison of the relative PEG-3 promoter luciferaseactivity in E11 and E11-NMT cells after transfection with an intact ratPEG-3 promoter (1) or various deletion mutants of the rat PEG-3 promoter(2 through 11). Further information in FIG. 24.

[0054]FIG. 28: Relative PEG-3 promoter luciferase activity in E11 cellstransformed by the PKC β_(l) gene (E11-PKC) after transfection with anintact rat PEG-3 promoter (1) or various deletion mutants of the ratPEG-3 promoter (2 through 11). Further information in FIG. 24.

[0055]FIG. 29: Relative PEG-3 promoter luciferase activity in CREF cellstransformed by human papilloma virus type 18 (CREF-HPV) aftertransfection with an intact rat PEG-3 promoter (1) or various deletionmutants of the rat PEG-3 promoter (2 through 11). Further information inFIG. 24.

[0056]FIG. 30: Relative PEG-3 promoter luciferase activity in CREF cellstransformed by the Ha-ras oncogene (CREF-ras) after transfection with anintact rat PEG-3 promoter (1) or various deletion mutants of the ratPEG-3 promoter (2 through 11). Further information in FIG. 24.

[0057]FIG. 31: Relative PEG-3 promoter luciferase activity in CREF-Trans6 cells transformed by human prostatic carcinoma (LNCaP) DNA (CREF-Trans6:4 NMT) after transfection with an intact rat PEG-3 promoter (1) orvarious deletion mutants of the rat PEG-3 promoter (2 through 11).Further information in FIG. 24.

[0058]FIG. 32: Schematic outline of CURE (Cancer Utilized ReporterExecution) strategy and construction of CIRAs (Cancer InhibitoryRecombinant Adenoviruses). The PEG-3 promoter is linked to variousgenes, including (1) wt-p53, (2) mda-7, (3) p21, (4) Ad-E1A, (5) HSV-TK,(6) ImStim (immunostimulatory gene, or (7) Antigen (molecule encoding animmunogenic molecule increasing tumor reactivity with immune cells). Thevarious constructs display slective gene expression as a function ofPEG-3 promoter activation, which is restricted to cancer cells. Thegenes are incorporated into a replication defective (1, 2, 3, 5, 6, and7) or replication competent (1, 2, 3, 4, 5, 6, and 7) adenovirus (Ad).These adenoviruses can then be used to infect human cancer cellsresulting in PEG-3 promoter activation of gene expression resulting intranscription of the linked gene and production of the encoded geneproduct. When normal cells are infected with any of the adenovirusconstructs (CIRAs), the promoter is inactive or marginally activeresulting in either no or small quantities of the encoded gene product.In these contexts, no physiological change should occur in normal cells.In contrast, when expressed in tumor cells the PEG-3 promoter is activeresulting in transcription of the linked gene and production of theencoded gene product. In the case of Ad 1, 2, and 3, infection of cancercells results in wt-p53, mda-7 or p21 protein. These proteins willresult in inhibition of cancer growth and in specific contexts willinduce programmed cell death (apoptosis). In the case of Ad 4, which isa replication competent Ad, induction of Ad E1A will result in viralreplication and lysis of the cancer cell. In the case of Ad 5, inductionof the HSV-TK gene renders the cell sensitive to growth inhibition andtoxicity following administration of gangcyclovir or acyclovir. In thecase of Ad 6, induction of an ImStim (Immunostimulatory gene), such asGM-CSF, IL-2, a cytokine (immune interferon, interleukin 6, etc.) or animmunomodulating protein, renders the cancer cell susceptible toimmunological attack and cell lysis. In the case of Ad 7, induction ofantigenic expression (with and without expression of co-stimulatorymolecules), renders the cancer cell susceptible to antibody mediatedtoxicity. This can result from an interaction with an antibody withdirect antitumor activity, an antibody linked to an immunotoxin, anantibody linked to a high energy radionuclide, etc. Ad 7 CIRAsexpressing proteins with T-cell epitopes or T-cell epitopes themselvescan be used to sensitize cancer cells to killing by CD8 cytotoxic killerT-cells. In principle, CIRAs can be produced that will result in thetargeted destruction of only cancer cells (the basis of the CUREtechnology). In addition to using CIRAs, CURE can also be used withalternative transfer systems, including a retrovirus, anadeno-associated virus, a herpes virus, a vaccinia virus, a liposomepreparation, physical delivery technology, naked DNA technology, etc.

DETAILED DESCRIPTION OF THE INVENTION

[0059] This invention provides an isolated nucleic acid moleculeencoding a Progression Elevated Gene-3 protein. The nucleic acid may beDNA, cDNA, genomic DNA or RNA.

[0060] This invention also encompasses DNAs and cDNAs which encode aminoacid sequences which differ from those of Progression Elevated Gene-3protein, but which should not produce phenotypic changes. Alternatively,this invention also encompasses DNAs and cDNAs which hybridize to theDNA and cDNA of the subject invention. Hybridization methods arewell-known to those of skill in the art.

[0061] The DNA molecules of the subject invention also include DNAmolecules coding for polypeptide analogs, fragments or derivatives ofantigenic polypeptides which differ from naturally-occurring forms interms of the identity or location of one or more amino acid residues(deletion analogs containing less than all of the residues specified forthe protein, substitution analogs wherein one or more residues specifiedare replaced by other residues and additional analogs where in one ormore amino acid residues is added to a terminal or medial portion of thepolypeptides) and which share some or all properties ofnaturally-occurring forms. These molecules include: the incorporation ofcodons “preferred” for expression by selected non-mammalian hosts; theprovision of sites for cleavage by restriction endonuclease enzymes; andthe provision of additional initial, terminal or intermediate DNAsequences that facilitate construction of readily expressed vectors.

[0062] The DNA molecules described and claimed herein are useful for theinformation which they provide concerning the amino acid sequence of thepolypeptide and as products for the large scale synthesis of thepolypeptide by a variety of recombinant techniques. The molecule isuseful for generating new cloning and expression vectors, transformedand transfected prokaryotic and eukaryotic host cells, and new anduseful methods for cultured growth of such host cells capable ofexpression of the polypeptide and related products.

[0063] Moreover, the isolated nucleic acid molecules encoding aProgression Elevated Gene-3 are useful for the development of probes tostudy the progression of cancer. This invention also provides isolatednucleic acid molecule encoding a human Progression Elevated Gene-3protein.

[0064] This invention provides a nucleic acid molecule of at least 12nucleotides capable of specifically recognizing a nucleic acid moleculeencoding a Progression Elevated Gene-3 protein. In a preferredembodiment, this nucleic acid molecule has a unique sequence of theProgression Elevated Gene-3. The unique sequence of the ProgressionElevated Gene-3 may easily be determined by comparing its sequence withknown sequences which are available in different databases. The nucleicacid molecule may be DNA or RNA.

[0065] This nucleic acid molecule of at least 15 nucleotides capable ofspecifically hybridizing with a sequence of a nucleic acid moleculeencoding a Progression Elevated Gene-3 protein can be used as a probe.Nucleic acid probe technology is well-known to those skilled in the artwho will readily appreciate that such probes may vary greatly in lengthand may be labeled with a detectable label, such as a radioisotope orfluorescent dye, to facilitate detection of the probe. DNA probemolecules may be produced by insertion of a DNA molecule which encodesProgression Elevated Gene-3 protein into suitable vectors, such asplasmids or bacteriophages, followed by transforming into suitablebacterial host cells, replication in the transformed bacterial hostcells and harvesting of the DNA probes, using methods well-known in theart. Alternatively, probes may be generated chemically from DNAsynthesizers.

[0066] RNA probes may be generated by inserting the Progression ElevatedGene-3 molecule downstream of a bacteriophage promoter such as T3, T7 orSP6. Large amounts of RNA probe may be produced by incubating thelabeled nucleotides with the linearized Progression Elevated Gene-3fragment where it contains an upstream promoter in the presence of theappropriate RNA polymerase.

[0067] This invention provides a method of detecting expression of theProgression Elevated Gene-3 in a sample which contains cells comprisingsteps of: (a) obtaining RNA from the cells; (b) contacting the RNA soobtained with a labelled probe of the Progression Elevated Gene-3 underhybridizing conditions permitting specific hybridization of the probeand the RNA; and (c) determining the presence of RNA hybridized to themolecule, thereby detecting the expression of the Progression ElevatedGene-3 in the sample. mRNA from the cell may be isolated by manyprocedures well-known to a person of ordinary skill in the art. Thehybridizing conditions of the labelled nucleic acid molecules may bedetermined by routine experimentation well-known in the art. Thepresence of mRNA hybridized to the probe may be determined by gelelectrophoresis or other methods known in the art. By measuring theamount of the hybrid made, the expression of the Progression ElevatedGene-3 protein by the cell can be determined. The labelling may beradioactive. For an example, one or more radioactive nucleotides can beincorporated in the nucleic acid when it is made.

[0068] The RNA obtained in step (a) may be amplified by polymerase chainreaction (PCR) with appropriate primers. The appropriate primers may beselected from the known Progression Elevated Gene-3 sequences. Insteadof detection by specific PEG-3 probe as described in the precedingparagraph, the specific amplified DNA by PCR is an indication that thereis expression of Progression Elevated Gene-3.

[0069] This invention provides an isolated nucleic acid moleculeencoding a Progression Elevated Gene-3 protein operatively linked to aregulatory element. In an embodiment, the vector is a plasmid.

[0070] This invention provides a host vector system for the productionof a polypeptide having the biological activity of a ProgressionElevated Gene-3 protein which comprises the vector having the sequenceof Progression Elevated Gene-3 and a suitable host. The suitable hostincludes but is not limited to a bacterial cell, yeast cell, insectcell, or animal cell.

[0071] The isolated Progression Elevated Gene-3 sequence can be linkedto different vector systems. Various vectors including plasmid vectors,cosmid vectors, bacteriophage vectors and other viruses are well-knownto ordinary skilled practitioners. This invention further provides avector which comprises the isolated nucleic acid molecule encoding forthe Progression Elevated Gene-3 protein.

[0072] As an example to obtain these vectors, insert and vector DNA canboth be exposed to a restriction enzyme to create complementary ends onboth molecules which base pair with each other and are then ligatedtogether with DNA ligase. Alternatively, linkers can be ligated to theinsert DNA which correspond to a restriction site in the vector DNA,which is then digested with the restriction enzyme which cuts at thatsite. Other means are also available and known to an ordinary skilledpractitioner.

[0073] In an embodiment, the rat PEG-3 sequence is cloned in the EcoRIsite of pZeoSV vector. This plasmid, pPEG-3, was deposited on Mar. 6,1997 with the American Type Culture Collection (ATCC), 12301 ParklawnDrive, Rockville, Md. 20852, U.S.A. under the provisions of the BudapestTreaty for the International Recognition of the Deposit of Microorganismfor the Purposes of Patent Procedure. Plasmid, pPEG-3, was accorded ATCCAccession Number 97911.

[0074] This invention further provides a host vector system for theproduction of a polypeptide having the biological activity of theProgression Elevated Gene-3 protein. These vectors may be transformedinto a suitable host cell to form a host cell vector system for theproduction of a polypeptide having the biological activity of theProgression Elevated Gene-3 protein.

[0075] Regulatory elements required for expression include promotersequences to bind RNA polymerase and transcription initiation sequencesfor ribosome binding. For example, a bacterial expression vectorincludes a promoter such as the lac promoter and for transcriptioninitiation the Shine-Dalgarno sequence and the start codon AUG.Similarly, a eukaryotic expression vector includes a heterologous orhomologous promoter for RNA polymerase II, a downstream polyadenylationsignal, the start codon AUG, and a termination codon for detachment ofthe ribosome. Such vectors may be obtained commercially or assembledfrom the sequences described by methods well-known in the art, forexample the methods described above for constructing vectors in general.Expression vectors are useful to produce cells that express theProgression Elevated Gene-3 protein.

[0076] This invention further provides an isolated DNA, cDNA or genomicDNA molecule described hereinabove wherein the host cell is selectedfrom the group consisting of bacterial cells (such as E. coli), yeastcells, fungal cells, insect cells and animal cells. Suitable animalcells include, but are not limited to Vero cells, HeLa cells, Cos cells,CV1 cells and various primary mammalian cells.

[0077] This invention further provides a method of producing apolypeptide having the biological activity of the Progression ElevatedGene-3 protein which comprising growing host cells of a vector systemcontaining Progression Elevated Gene-3 sequence under suitableconditions permitting production of the polypeptide and recovering thepolypeptide so produced.

[0078] This invention provides a mammalian cell comprising a DNAmolecule encoding a Progression Elevated Gene-3 protein, such as amammalian cell comprising a plasmid adapted for expression in amammalian cell, which comprises a DNA molecule encoding a ProgressionElevated Gene-3 protein and the regulatory elements necessary forexpression of the DNA in the mammalian cell so located relative to theDNA encoding the Progression Elevated Gene-3 protein as to permitexpression thereof.

[0079] Various mammalian cells may be used as hosts, including, but notlimited to, the mouse fibroblast cell NIH3T3, CHO cells, HeLa cells,Ltk⁻ cells, Cos cells, etc. Expression plasmids such as that describedsupra may be used to transfect mammalian cells by methods well-known inthe art such as calcium phosphate precipitation, electroporation or DNAencoding the Progression Elevated Gene-3 protein may be otherwiseintroduced into mammalian cells, e.g., by microinjection, to obtainmammalian cells which comprise DNA, e.g., cDNA or a plasmid, encoding aProgression Elevated Gene-3 protein.

[0080] This invention also provides a purified Progression ElevatedGene-3 protein and a fragment thereof. As used herein, the term“purified Progression Elevated Gene-3 protein” shall mean isolatednaturally-occurring Progression Elevated Gene-3 protein or proteinmanufactured such that the primary, secondary and tertiary conformation,and posttranslational modifications are identical to naturally-occurringmaterial as well as non-naturally occurring polypeptides having aprimary structural conformation (i.e. continuous sequence of amino acidresidues). Such polypeptides include derivatives and analogs. Thefragment should bear biological activity similar to the full-lengthProgression Elevated Gene-3 protein.

[0081] This invention also provides a polypeptide encoded by theisolated vertebrate nucleic acid molecule having a sequence of aProgression Elevated Gene-3.

[0082] This invention provides an antibody capable of specificallybinding to a Progression Elevated Gene-3 protein. The antibody may bepolyclonal or monoclonal. This invention provides a method to selectspecific regions on the Progression Elevated Gene-3 to generateantibodies. The protein sequence may be determined from the DNAsequence. The hydrophobic or hydrophilic regions in the protein will beidentified. Usually, the hydrophilic regions will be more immunogenicthan the hydrophobic regions. Therefore the hydrophilic amino acidsequences may be selected and used to generate antibodies specific tothe Progression Elevated Gene-3 protein.

[0083] Polyclonal antibodies against these peptides may be produced byimmunizing animals using the selected peptides. Monoclonal antibodiesare prepared using hybridoma technology by fusing antibody producing Bcells from immunized animals with myeloma cells and selecting theresulting hybridoma cell line producing the desired antibody.Alternatively, monoclonal antibodies may be produced by in vitrotechniques known to a person of ordinary skill in the art. Specificantibody which only recognizes the Progression Elevated Gene-3 proteinwill then be selected. The selected antibody is useful to detect theexpression of the Progression Elevated Gene-3 in living animals, inhumans, or in biological tissues or fluids isolated from animals orhumans.

[0084] This invention provides a method of transforming cells whichcomprises transfecting a host cell with a suitable vector having thesequence of a Progression Elevated Gene-3. This invention also providesthe transformed cells produced by this method.

[0085] This invention provides a method for determining whether cellsare in progression comprising steps of: a) measuring the expression ofthe Progression Elevated Gene-3; and b) comparing the expressionmeasured in step a) with the expression of Progression Elevated Gene-3in cells which are known not to be in progression, wherein an increaseof the expression indicates that the cells are in progression. In anembodiment, the expression of Progression Elevated Gene-3 is measured bythe amount of Progression Elevated Gene-3 mRNA expressed in the cells.In another embodiment, the expression of Progression Elevated Gene-3 ismeasured by the amount of the Progression Elevated Gene-3 proteinexpressed in the cells.

[0086] This invention provides a method for determining whether a cancercell is in a progression stage comprising measuring the expression ofProgression Elevated Gene-3 in the cancer cell, wherein an increase inthe amount indicates that the cancer cell is in progression.

[0087] This invention provides a method for diagnosing theaggressiveness of cancer cells comprising measuring the expression ofProgression Elevated Gene-3 in the cancer cell, wherein an increase inthe amount of the expression indicates that the cancer cell is moreaggressive.

[0088] This invention provides a pharmaceutical composition forreversing the progression state of cells comprising an amount of thenucleic acid molecule capable of specifically hybridizing theProgression Elevated Gene-3 protein effective to inhibit the expressionof Progression Elevated Gene-3 and a pharmaceutically acceptablecarrier.

[0089] Pharmaceutically acceptable carriers are well-known to thoseskilled in the art. Such pharmaceutically acceptable carriers may beaqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, saline and buffered media.Parenteral vehicles include sodium chloride solution, Ringer's dextrose,dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present, suchas, for example, antimicrobials, antioxidants, chelating agents, inertgases and the like.

[0090] This invention provides a pharmaceutical composition forreversing the progression state of cells comprising an amount of theantibody or a functional fragment thereof which is capable ofspecifically recognizing the Progression Elevated Gene-3 proteineffective to neutralize the action of the Progression Elevated Gene-3protein and a pharmaceutically acceptable carrier.

[0091] This invention provides a method for producing cells which areresistant to progression comprising inhibiting or eliminating theexpression of Progression Elevated Gene-3 in the cells. This inventionalso provides cells resulting from the method.

[0092] This invention provides a method for protecting cells fromtherapeutic damage comprising inhibiting or eliminating the expressionof Progression Elevated Gene-3 in the cells. In an embodiment, thedamage is resulted from chemotherapy. In another embodiment, the damageis resulted from physical agent. Such physical agent includes but is notlimited to gamma-irradiation.

[0093] One method to inhibit the expression of Progression ElevatedGene-3 is by expression of effective amount antisense RNA in the cellthereby inhibiting the expression of Progression Elevated Gene-3. Theexpression of Progression Elevated Gene-3 may be eliminated by deletionof the gene or introduction of mutation(s) to the gene.

[0094] This invention provides a transgenic nonhuman living organismexpressing Progression Elevated Gene-3 protein. In an embodiment, theliving organism is animal.

[0095] One means available for producing a transgenic animal, with amouse as an example, is as follows: Female mice are mated, and theresulting fertilized eggs are dissected out of their oviducts. The eggsare stored in an appropriate medium. DNA or cDNA encoding a ProgressionElevated Gene-3 is purified from a vector by methods well-known in theart. Inducible promoters may be fused with the coding region of the DNAto provide an experimental means to regulate expression of thetrans-gene. Alternatively or in addition, tissue specific regulatoryelements may be fused with the coding region to permit tissue-specificexpression of the trans-gene. The DNA, in an appropriately bufferedsolution, is put into a microinjection needle (which may be made fromcapillary tubing using a pipet puller) and the egg to be injected is putin a depression slide. The needle is inserted into the pronucleus of theegg, and the DNA solution is injected. The injected egg is thentransferred into the oviduct of a pseudopregnant mouse (a mousestimulated by the appropriate hormones to maintain pregnancy but whichis not actually pregnant), where it proceeds to the uterus, implants,and develops to term. As noted above, microinjection is not the onlymethod for inserting DNA into the egg cell, and is used here only forexemplary purposes.

[0096] This invention provides a cell having an exogenous indicator geneunder the control of the regulatory element of a Progression ElevatedGene-3. In an embodiment, the cell is at progression. This cell may beproduced by introducing an indicator gene to an E11-NMT, CREF-ras orCREF-src cell.

[0097] In a separate embodiment, the cell having an exogenous indicatorgene under the control of the regulatory element of a ProgressionElevated Gene-3 is not at progression. This cell may be produced byintroducing an indicator gene to the E11 or the CREF cell.

[0098] The indicator gene codes for beta-galactosidase, luciferase,chloramphenicol transferase or secreted alkaline phosphatase. Otherindicator gene such as the Green Fluorescent Protein gene may be similarused in this invention. The indicator provides an easily detectablesignal when the PEG-3 is expressed.

[0099] This invention provides a method for determining whether an agentis capable of inhibiting DNA damage and repair pathways, cancerprogression or oncogene mediated transformation comprising contacting anamount of the agent with the cell having an exogenous indicator geneunder the control of the regulatory element of a Progression ElevatedGene-3, wherein a decrease of expression of the indicator gene indicatesthat the agent is capable of inhibiting DNA damage and repair pathways,cancer progression or oncogene mediated transformation. This inventionprovides a method for determining whether an agent is capable ofinducing DNA damage and repair pathways, cancer progression or oncogenemediated transformation comprising contacting an amount of the agentwith the cell having an exogenous indicator gene under the control ofthe regulatory element of a Progression Elevated Gene-3 is not atprogression, wherein an increase of expression of the indicator geneafter the contact indicates that the agent is capable of inducing DNAdamage and repair pathways, cancer progression or oncogene mediatedtransformation.

[0100] Large scale of agents may be screened by the above two methodsthrough automation. Indicator gene which produces color reaction may beselected.

[0101] This invention provides a cell having an exogenous suicidal geneor genes under the control of the regulatory element of a ProgressionElevated Gene-3. Such “suicidal gene” will disrupt the normal progressof the cell. Preferably, the switching on of the suicidal gene will leadto cell death or halt in cell growth. Example of such genes are geneswhich lead to apotosis.

[0102] This invention provides a nucleic acid molecule comprising asequence of the promoter of a Progression Elevated Gene-3 protein.

[0103] This invention also provides a nucleic acid molecule comprisingCis-Acting Regulatory Elements of the promoter of a Progression ElevatedGene-3 protein.

[0104] This invention also provides a Trans-Acting Regulatory Elementthat activates the expression of Progression Elevated Gene-3.

[0105] This invention further provides Trans-Acting Regulatory Elementthat suppresses the expression of Progression Elevated Gene-3.

[0106] This invention also provide an isolated nucleic acid moleculecomprising sequence encoding the Trans-Acting Regulatory Element.

[0107] This invention provides an isolated nucleic acid moleculeencoding a Progression Elevated Gene-3 protein. This invention alsoprovides the above-described nucleic acid, wherein the nucleic acidencodes a human Progression Elevated Gene-3 protein.

[0108] In addition, this invention provides the above-described nucleicacid, wherein the nucleic acid encodes a rodent Progression ElevatedGene-3 protein.

[0109] This invention further provides an isolated nucleic acidcomprising substantially the same sequence as the sequence set forth inFIG. 11 or 13 or the complement of the sequence set forth in FIG. 11 or13.

[0110] This invention also provides an isolated nucleic acid sequencecomprising a nucleic acid sequence that specifically hybridizes to thesequence set forth in FIG. 11 or 13 or the complement of the sequenceset forth in FIG. 11 or 13.

[0111] This invention provides an isolated nucleic acid comprisingsubstantially the same sequence as the nucleic acid sequence encodingthe 80 C-terminal-most amino acids, wherein the nucleic acid sequence isset forth in FIG. 11 or 13 or the complement of the sequence set forthin FIG. 11 or 13.

[0112] This invention also provides an isolated nucleic acid comprisingsubstantially the same sequence as the nucleic acid sequence encodingthe 80 C-terminal-most amino acids, wherein the nucleic acid sequence isset forth in FIG. 11 or 13 or the complement of the sequence set forthin FIG. 11 or 13.

[0113] This invention also provides an isolated nucleic acid sequencecomprising a nucleic acid sequence that specifically hybridizes to thenucleic acid sequence encoding the 80 C-terminal-most amino acids,wherein the nucleic acid sequence is set forth in FIG. 11 or 13 or thecomplement of the sequence set forth in FIG. 11 or 13.

[0114] This invention further provides the above-described nucleic acid,wherein the nucleic acid is DNA, cDNA, genomic DNA, or RNA.

[0115] This invention provides a nucleic acid molecule comprising apromoter of Progression Elevated Gene-3.

[0116] This invention also provides a nucleic acid molecule comprisingcis-acting regulatory element of Progression Elevated Gene-3 protein.

[0117] In addition, this invention provides a trans-acting regulatoryelement that activates the expression of Progression Elevated Gene-3.

[0118] This invention also provides a trans-acting regulatory elementthat suppresses the expression of Progression Elevated Gene-3.

[0119] This invention further provides an isolated nucleic acid moleculecomprising sequence encoding the trans-acting regulatory element ofclaim 12 or 13.

[0120] This invention provides a purified Progression Elevated Gene-3protein.

[0121] This invention provides the polypeptide encoded by theabove-described nucleic acids.

[0122] This invention also provides an isolated polypeptide comprisingsubstantially the same sequence as the sequence set forth in FIG. 12 or13.

[0123] This invention provides the above-described polypeptide, whereinthe polypeptide has tumor progression activity or the presence of thepolypeptide positively correlates with the progression phenotype.

[0124] In addition, this invention also provides an isolated polypeptidecomprising substantially the same sequence as the sequence of the 80C-terminal-most amino acids set forth in FIG. 12 or 13 or the complementof the sequence set forth in FIG. 12 or 13.

[0125] This invention provides a nucleic acid molecule comprising 12 ormore nucleotides that specifically hybridize with the above-describednucleic acids.

[0126] This invention also provides an antisense polynucleotidecomprising a sequence complementary to the above-described nucleicacids.

[0127] This invention further provides an upstream nucleic acid sequencecomprising nucleotides 1-500 as set forth in FIG. 14.

[0128] In addition, this invention provides an upstream nucleic acidsequence comprising nucleotides 1-1000 as set forth in FIG. 14.

[0129] This invention provides an upstream nucleic acid sequencecomprising the nucleic acid sequence set forth in FIG. 14.

[0130] This invention also provides an antisense nucleic acid comprising15 or more nucleotides capable of specifically hybridizing to theabove-described upstream nucleic acid sequences.

[0131] This invention further provides an antisense nucleic acidcomprising 15 or more nucleotides that specifically hybridizes to thenucleotides 1-500 as set forth in FIG. 14.

[0132] This invention provides an isolated polypeptide comprising atleast a portion of a progression-associated protein, or a variantthereof, wherein: a) the progression-associated protein comprises asequence encoded by a nucleotide sequence set forth in FIG. 11 or 13;and b) the portion retains at least one immunological or biologicalactivity characteristic of the progression-associated protein.

[0133] This invention provides the above-described polypeptides, whereinthe portion is immunologically active.

[0134] This invention also provides an isolated nucleic encoding theabove-described polypeptides.

[0135] This invention provides a vector which comprises theabove-described isolated nucleic acids.

[0136] This invention also provides the above-described isolated nucleicacids operatively linked to a regulatory element.

[0137] This invention further provides the above-described vector,wherein the vector is a plasmid.

[0138] This invention also provides the above-described plasmid,designated PGEN-3 (ATCC Accession No. 97911).

[0139] This invention provides a host vector system for the productionof a polypeptide having the biological activity of a ProgressionElevated Gene-3 protein which comprises the above-described vectors anda suitable host.

[0140] This invention further provides the above-described vectors,wherein the suitable host is a bacterial cell, yeast cell, insect cell,or animal cell.

[0141] In addition, this invention also provides an expression vectorcomprising the above-described nucleic acids.

[0142] This invention provides a host cell transformed or transfectedwith the above-described expression vectors.

[0143] This invention further provides an antibody or antigen-bindingfragment thereof that specifically binds to the above-describedpolypeptides.

[0144] This invention provides a kit for inhibiting tumor progression,comprising an antisense nucleic acid capable of specifically hybridizingto the above-described nucleic acids.

[0145] This invention provides the above-described kit, wherein theantisense nucleic acid is linked to a promoter.

[0146] This invention further provides the above-described kits, whereinthe antisense nucleic acid linked to a promoter is part of an expressionvector.

[0147] In addition, this invention provides the above-described kits,wherein the expression vector is adapted for expression in a mammaliancell.

[0148] This invention further provides a method of detecting expressionof the Progression Elevated Gene-3 in a sample which contains cellscomprising steps of: a) obtaining RNA from the cells; b) contacting thenucleic acid so obtained with a labeled form of the above-describednucleic acids under hybridizing conditions; and c) detecting thepresence of RNA hybridized to the molecule, thereby detecting theexpression of the Progression Elevated Gene-3 in the sample.

[0149] This invention also provides the above-described method, furthercomprising amplification of the RNA obtained in step a prior to thecontacting of step b.

[0150] In addition, this invention provides the above-described methods,wherein the expression of Progression Elevated Gene-3 is measured by theamount of Progression Elevated Gene-3 mRNA expressed in the cells.

[0151] This invention provides the above-described methods, wherein theexpression of Progression Elevated Gene-3 is measured by the amount ofthe Progression Elevated Gene-3 protein expressed in the cells.

[0152] This invention provides a method for determining whether a cancercell is in a progression stage comprising measuring the expression ofProgression Elevated Gene-3 in the cancer cell, wherein an increase inthe amount indicates that the cancer cell is in progression.

[0153] This invention provides a method for diagnosing theaggressiveness of cancer cells comprising measuring the expression ofProgression Elevated Gene-3 in the cancer cell, wherein an increase inthe amount of the expression indicates that the cancer cell is moreaggressive.

[0154] This invention further provides a method of monitoring tumorprogression in a subject, comprising: a) obtaining at least one nucleicacid sample from the subject; and b) determining the quantity of theabove-described nucleic acids in the nucleic acid sample.

[0155] This invention provides a method of monitoring DNA damage in asubject, comprising: a) obtaining at least one nucleic acid sample fromthe subject; and b) determining the quantity of the above-describednucleic acids in the nucleic acid sample.

[0156] In addition, this invention provides the above-described methods,wherein the quantity of nucleic acid positively correlates withtransformation progression.

[0157] This invention also provides the above-described methods, whereintwo or more nucleic acid samples are taken from the subject at differenttimes.

[0158] This invention also provides the above-described methods, furthercomprising using the nucleic acid samples in determining differences inthe expression of the above-described nucleic acids over time.

[0159] This invention further provides a kit for diagnosing tumorprogression, comprising a nucleic acid consisting of a sequence of 15 ormore nucleotides that specifically hybridizes to the above-describednucleic acids.

[0160] In addition, this invention also provides a kit for diagnosingtumor progression, comprising an antibody to the above-describedpolypeptides.

[0161] This invention provides a method for determining whether cellsare in progression, comprising the steps of: a) measuring expression ofPEG-3 in a sample of cells; and b) comparing the expression measured instep a with the expression of PEG-3 in cells that are not inprogression, thereby determining whether the cells are in progression.

[0162] This invention further provides a method for determining whethera cancer in a patient is in progression, comprising detecting in abiological sample obtained from the patient the above-describedpolypeptides, thereby determining whether a cancer in the patient is inprogression.

[0163] A method for determining whether a cancer in a patient is inprogression, comprising detecting, in a biological sample obtained fromthe patient, a nucleic acid encoding the above-described polypeptides ora portion thereof, thereby determining whether a cancer in the patientis in progression.

[0164] This invention provides the above-described methods, wherein thedetecting comprises: preparing cDNA from RNA molecules in the biologicalsample; and specifically amplifying cDNA molecules encoding at least aportion of the above-described polypeptides.

[0165] This invention also provides a method for monitoring theprogression of a cancer in a patient, comprising: a) detecting, in abiological sample obtained from a patient, the above-describedpolypeptides at a first point in time; b) repeating step (a) at asubsequent point in time; and c) comparing the amounts of polypeptidedetected in steps a and b, thereby monitoring the progression of acancer in the patient.

[0166] This invention further provides the above-described methods,wherein the step of detecting comprises contacting a portion of thebiological sample with a monoclonal antibody that specificallyrecognizes the above-described polypeptides.

[0167] This invention provides the above-described methods, wherein thebiological sample is a portion of a tumor.

[0168] This invention provides a method for monitoring the progressionof a cancer in a patient, comprising: a) detecting, in a biologicalsample obtained from a patient, an amount of an RNA molecule encodingthe above-described polypeptides at a first point in time; b) repeatingstep a at a subsequent point in time; and c) comparing the amounts ofRNA molecules detected in steps a and b, thereby monitoring theprogression of a cancer in the patient.

[0169] This invention also provides a diagnostic kit, comprising: a) theabove-described antibody or fragment thereof; and b) a second antibodyor fragment thereof that binds to (i) the monoclonal antibody recited instep a; or (ii) the above-described polypeptide; wherein the secondmonoclonal antibody is conjugated to a reporter group.

[0170] This invention provides a cell comprising an exogenous indicatorgene under the control of the regulatory element of a ProgressionElevated Gene-3.

[0171] This invention further provides above-described cell, wherein thecell is an E11 or CRF.

[0172] This invention further provides above-described cell, wherein thecell is not at progression.

[0173] In addition, this invention provides above-described cell,wherein the cell is an E11-NMT, CREF-ras, CREF-HPV, or CREF-src cell.

[0174] This invention also provides above-described cell, wherein thecell is at progression.

[0175] This invention provides above-described cell, wherein theindicator gene encodes beta-galactosidase, luciferase, chloramphenicoltransferase or a secreted alkaline phosphatase.

[0176] This invention further provides a method for determining whetheran agent is capable of inhibiting DNA damage and repair pathways, cancerprogression or oncogene mediated transformation, comprising contactingthe agent with the above-described cells, wherein a decrease ofexpression of the indicator gene indicates that the agent is capable ofinhibiting DNA damage and repair pathways, cancer progression oroncogene mediated transformation.

[0177] This invention also provides a method for determining whether anagent is capable of inducing DNA damage and repair pathways, cancerprogression or oncogene mediated transformation, comprising contactingthe agent with the above-described cells, wherein an increase ofexpression of the indicator gene after the contact indicates that theagent is capable of inducing DNA damage and repair pathways, cancerprogression or oncogene mediated transformation.

[0178] This invention provides a method for identifying an agent thatmodulates the expression of PEG-3, comprising: a) contacting a candidateagent with a cell transformed or transfected with a reporter gene underthe control of a PEG-3 promoter or a regulatory element thereof underconditions and for a time sufficient to allow the candidate agent todirectly or indirectly alter expression of the promoter or regulatoryelement thereof; and b) determining the effect of the candidate agent onthe level of reporter protein produced by the cell, thereby identifyingan agent that modulates expression of PEG-3.

[0179] This invention also provides the above-described method, whereinthe agent activates the PEG-3 promoter indirectly by interacting with anoncogene.

[0180] This invention further provides a method for identifying an agentthat modulates the ability of PEG-3 to induce progression, comprising:a) contacting a candidate agent with the above-described polypeptides,under conditions and for a time sufficient to allow the candidate agentand polypeptide to interact; and b) determining the effect of thecandidate agent on the ability of the polypeptide to induce progression,thereby identifying an agent that modulates the ability of PEG-3 toinduce progression.

[0181] This invention also provides a cell comprising theabove-described nucleic acids encoding PEG-3 linked to a tissue specificpromoter.

[0182] This invention provides a cell comprising a reporter gene linkedto a PEG-3 promoter.

[0183] In addition, this invention provides the above-described cells,wherein the reporter gene encodes luciferase or beta galactosidase.

[0184] This invention further provides the above-described cell, whereinthe cell comprises 4NMT or tumorigenic CREF-Trans 6 cells.

[0185] This invention provides the above-described cell, wherein thecell comprises CREF-ras, CREF-src, or CREF-HPV cells.

[0186] This invention further provides the above-described cell, whereinthe reporter gene encodes a cell surface protein.

[0187] This invention provides the above-described cell, wherein thecell surface protein is in a position accessible for binding to anantibody.

[0188] This invention provides a transgenic animal, comprising theabove-described cells.

[0189] This invention further provides the use of the above-describedcells to identify compounds that induce DNA damage.

[0190] This invention also provides the use of the above-described cellsto identify compounds that induce cancer progression.

[0191] This invention provides the use of the above-described cells toidentify compounds that induce oncogenic transformation.

[0192] In addition, this invention provides the use of theabove-described cells to identify compounds that induce or inhibitangiogenesis.

[0193] This invention further provides a method of identifying compoundsthat induce oncogenic transformation, comprising: exposing theabove-described cells to the compound and identifying compounds thatactivate the PEG-3 promoter.

[0194] This invention further provides a method of identifying compoundsthat induce DNA damage, comprising: exposing the above-described cellsto the compound and identifying compounds that activate the PEG-3promoter.

[0195] This invention provides a method of identifying compounds thatregulate angiogenesis, comprising: exposing the above-described cells tothe compound and identifying compounds that affect the activity of thePEG-3 promoter.

[0196] This invention provides above-described method, wherein theactivity of the PEG-3 promoter is monitor by assessing the level ofexpression of the reporter gene.

[0197] This invention further provides above-described method, whereincells are plated in microtiter plates for rapid screening.

[0198] This invention provides above-described method, wherein the cellis obtained from a transgenic animal and exposed to the compound invitro.

[0199] This invention also provides above-described method, wherein thecell is obtained by transfection or transformation and exposed to thecompound in vitro.

[0200] This invention further provides a method of identifying compoundsthat induce oncogenic transformation, comprising: exposing theabove-described transgenic animal to the compound and identifyingcompounds that activate the PEG-3 promoter.

[0201] In addition, this invention provides a method of identifyingcompounds that induce DNA damage, comprising: exposing theabove-described transgenic animal to the compound and identifyingcompounds that activate the PEG-3 promoter.

[0202] This invention further provides a method of identifying compoundsthat regulate angiogenesis, comprising: exposing the above-describedtransgenic animal to the compound and identifying compounds that affectthe activity of the PEG-3 promoter.

[0203] This invention provides the above-described methods, wherein theactivity of the PEG-3 promoter is monitor by assessing the level ofexpression of the reporter gene.

[0204] This invention also provides a method of producing a ProgressionElevated Gene-3 protein which comprises growing the above-describedvector under conditions permitting production of the protein andrecovering the protein so produced.

[0205] This invention further provides a pharmaceutical composition forreversing the progression state of cells comprising an amount of theabove-described nucleic acids effective to inhibit the expression ofProgression Elevated Gene-3 and a pharmaceutically acceptable carrier.

[0206] This invention provides a pharmaceutical composition forreversing the progression state of cells comprising an amount of theabove-described antibody or a functional fragment thereof effective toneutralize the action of the Progression Elevated Gene-3 protein and apharmaceutically acceptable carrier.

[0207] This invention further provides a method for producing cellswhich are resistant to progression comprising inhibiting or eliminatingthe expression of Progression Elevated Gene-3 in the cells.

[0208] In addition, this invention provides the cells resulting from theabove-described methods.

[0209] This invention provides a transgenic nonhuman living organismexpressing the above-described polypeptides.

[0210] In addition, this invention provides above-described transgenic,wherein the organism is an animal.

[0211] This invention further provides a pharmaceutical composition,comprising: a) the above-described polypeptides; and b) aphysiologically acceptable carrier.

[0212] This invention provides a vaccine, comprising: a) theabove-described polypeptides; and b) an immune response enhancer.

[0213] This invention further provides a pharmaceutical composition,comprising: a) the above-described nucleic acids; and b) aphysiologically acceptable carrier.

[0214] This invention provides a pharmaceutical composition, comprising:a) the above-described antibody; and b) a physiologically acceptablecarrier.

[0215] This invention further provides a method for inhibiting theprogression of a cancer in a subject, comprising administering to thesubject an agent that inhibits expression of PGEN-3.

[0216] This invention provides the above-described methods, whereinPGEN-3 is one of the above-described polypeptides.

[0217] This invention provides the above-described methods, whereinagent is one of the above-described nucleic acids.

[0218] In addition, this invention provides a method for preparing theabove-described polypeptides, comprising the steps of: a) culturing oneof the above-described host cells under conditions suitable for theexpression of the polypeptide; and b) recovering the polypeptide fromthe host cell culture.

[0219] This invention further provides a method for producing cells thatare resistant to progression, comprising inhibiting or eliminating theexpression of a PEG-3 gene in the cells.

[0220] This invention further provides a method for protecting cellsfrom chemotherapeutic damage, comprising inhibiting or eliminating theexpression of PEG-3 in the cells.

[0221] This invention provides a cell transformed or transfected with areporter gene under the control of a human PEG-3 promoter or regulatoryelement thereof.

[0222] This invention provides the above-described polypeptides, whereinthe progression phenotype comprises anchorage-independent growth,tumorigenesis, angiogenesis, or metastasis.

[0223] This invention further provides the above-described methods,wherein the cancer is melanoma.

[0224] This invention provides the above-described methods, wherein thecancer is brain cancer.

[0225] This invention further provides the above-described methods,wherein the cancer is cervical cancer.

[0226] This invention provides the above-described methods, wherein thecancer is prostate cancer.

[0227] In addition, this invention provides the above-described methods,wherein the cancer is breast cancer.

[0228] This invention further provides the above-described methods,wherein the cancer is nasal pharyngeal cancer.

[0229] In addition, this invention provides the above-described methods,wherein the cancer is neoblastoma multiforme cancer.

[0230] Methods to introduce a nucleic acid molecule into cells have beenwell known in the art. Naked nucleic acid molecule may be introducedinto the cell by direct transformation. Alternatively, the nucleic acidmolecule may be embedded in liposomes. Accordingly, this inventionprovides the above methods wherein the nucleic acid is introduced intothe cells by naked DNA technology, adenovirus vector, adeno-associatedvirus vector, Epstein-Barr virus vector, Herpes virus vector, attenuatedHIV vector, retroviral vectors, vaccinia virus vector, liposomes,antibody-coated liposomes, mechanical or electrical means. The aboverecited methods are merely served as examples for feasible means ofintroduction of the nucleic acid into cells. Other methods known may bealso be used in this invention.

[0231] This invention further provides the above-described methods,wherein the cancer is melanoma.

[0232] This invention provides the above-described methods, wherein thecancer is epithelial cancer.

[0233] This invention provides the above-described methods, wherein theepithelial cancer is brain, breast, cervical, prostate, lung orcolorectal cancer.

[0234] In addition, this invention further provides the above-describedmethods, wherein the cancer is derived from a central nervous systemtumor.

[0235] This invention provides the above-described methods, wherein thecentral nervous system tumor comprises a neuroblastoma or glioblastomacancer.

[0236] This invention further provides the above-described methods,wherein the cell comprises an endothelial cells.

[0237] In addition, this invention provides the above-described methods,wherein endothelial cell growth or proliferation is induced.

[0238] This invention further provides the above-described methods,wherein endothelial cell growth or proliferation is inhibited.

[0239] This invention provides an inducible PEG-3 regulatory regionfunctionally linked to a gene encoding a product that causes or may beinduced to cause the death or inhibition of cancer cells.

[0240] This invention further provides an inducible PEG-3 regulatoryregion functionally linked to a gene encoding a product that causes ormay be induced to cause the death or inhibition of cancer cell growth.

[0241] This invention also provides a vector suitable for introductioninto a cell, comprising: a) an inducible PEG-3 regulatory region; and b)a gene encoding a product that causes or may be induced to cause thedeath or inhibition of cancer cell growth.

[0242] Numerous vectors for expressing the inventive proteins may beemployed. Such vectors, including plasmid vectors, cosmid vectors,bacteriophage vectors and other viruses, are well known in the art. Forexample, one class of vectors utilizes DNA elements which are derivedfrom animal viruses such as bovine papilloma virus, polyoma virus,adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV orMOMLV), Semliki Forest virus or SV40 virus. Additionally, cells whichhave stably integrated the DNA into their chromosomes may be selected byintroducing one or more markers which allow for the selection oftransfected host cells. The markers may provide, for example,prototrophy to an auxotrophic host, biocide resistance or resistance toheavy metals such as copper. The selectable marker gene can be eitherdirectly linked to the DNA sequences to be expressed, or introduced intothe same cell by cotransformation.

[0243] Regulatory elements required for expression include promotersequences to bind RNA polymerase and transcription initiation sequencesfor ribosome binding. Additional elements may also be needed for optimalsynthesis of mRNA. These additional elements may include splice signals,as well as enhancers and termination signals. For example, a bacterialexpression vector includes a promoter such as the lac promoter and fortranscription initiation the Shine-Dalgarno sequence and the start codonAUG. Similarly, a eukaryotic expression vector includes a heterologousor homologous promoter for RNA polymerase II, a downstreampolyadenylation signal, the start codon AUG, and a termination codon fordetachment of the ribosome. Such vectors may be obtained commercially orassembled from the sequences described by methods well known in the art,for example the methods described above for constructing vectors ingeneral.

[0244] These vectors may be introduced into a suitable host cell to forma host vector system for producing the inventive proteins. Methods ofmaking host vector systems are well known to those skilled in the art.

[0245] Suitable host cells include, but are not limited to, bacterialcells (including gram positive cells), yeast cells, fungal cells, insectcells and animal cells. Suitable animal cells include, but are notlimited to HeLa cells, Cos cells, CvI cells and various primarymammalian cells. Numerous mammalian cells may be used as hosts,including, but not limited to, the mouse fibroblast cell N1H-3T3 cells,CHO cells, HeLa cells, Ltk⁻ cells and COS cells. Mammalian cells may betransfected by methods well known in the art such as calcium phosphateprecipitation, electroporation and microinjection.

[0246] In an embodiment, inducible promoters may be fused with thecoding region of the DNA to provide an experimental means to regulateexpression. Alternatively or in addition, tissue specific regulatoryelements may be fused with the coding region to permit tissue-specificexpression.

[0247] In addition, this invention further provides the above-describedvectors, wherein the inducible PEG-3 regulatory region is a promoter.

[0248] This invention also provides the above-described vectors, whereinthe inducible PEG-3 regulatory region is an enhancer.

[0249] This invention further provides the above-described vectors,wherein the gene encodes an inducer of apoptosis.

[0250] In addition, this invention provides the above-described vectors,wherein the gene is a tumor suppressor gene.

[0251] In an embodiment, tumor suppressors include agents that inhibittumor growth. In another embodiment, tumor suppressors include agentsthat inhibit, reverse, or reduce the cancer progression phenotype.

[0252] This invention also provides the above-described vectors, whereinthe tumor suppressor gene is p53.

[0253] In addition, this invention provides the above-described vectors,wherein the tumor suppressor gene is mda-7.

[0254] This invention also provides the above-described vectors, whereinthe tumor suppressor gene is p21.

[0255] In addition, this invention provides the above-described vectorswherein the gene encodes a viral replication protein.

[0256] This invention further provides the above-described vectors,wherein the gene is E1A.

[0257] This invention also provides the above-described vectors, whereinthe gene is E1B.

[0258] This invention also further provides the above-described vectors,wherein the gene encodes a product toxic to cells or an intermediateto-a product toxic to cells.

[0259] In an embodiment, products toxic to cells include chemicals thatreduce a cells chances and/or duration of survival. In an embodiment,the products toxic to cells are radioactive. In another embodiment, theproducts toxic to cells induce DNA damage. In another embodiment, theproducts toxic to cells inhibit a critical enzyme or regulatory protein.In another embodiment, the products toxic to cells contain or inducefree radicals. One skilled in the art would recognize a vast array ofother products that are toxic to cells.

[0260] Further, this invention provides the above-described vectors,wherein the gene encodes thymidine kinase.

[0261] In addition, this invention provides the above-described vectors,wherein the gene encodes a product causing enhanced immune recognitionof the cell.

[0262] Further, this invention provides the above-described vectors,wherein the gene is GM-CSF.

[0263] This invention also provides the above-described vectors, whereinthe gene is IL-2.

[0264] This invention also provides the above-described vector, whereinthe gene encodes a cytokine.

[0265] Further, this invention provides the above-described vector,wherein the cytokine is IF-gamma.

[0266] In addition, this invention provides the above-described vector,wherein the cytokine is IL-6.

[0267] This invention also provides the above-described vector, whereinthe gene encodes an immunomodulator.

[0268] Further, this invention provides the above-described vector,wherein the gene encodes a T-cell epitope.

[0269] This invention also provides the above-described vector, whereinthe gene encodes a T-cell reactive protein.

[0270] This invention further provides the above-described vectors,wherein the gene encodes a product causing the cell to express aspecific antigen.

[0271] In an embodiment, the gene causes the cells to express an antigenon their surface; thus, allowing the cells to be targeted by antibodiesspecific to the antigen.

[0272] This invention also provides a method of treating cancer in asubject, comprising: a) administering the one or more of theabove-described vectors to the subject; and b) administering gancycloviror acyclovir to the subject.

[0273] In addition, this invention provides a method of treating cancerin a subject, comprising: a) administering one of the above-describedvectors to the subject; and b) administering an antibody or a fragmentof an antibody to the the above-described antigen to the subject.

[0274] Further, this invention provides the above-described methods,wherein the antibody is toxic or linked to a toxic substance.

[0275] This invention also provides the above-described methods, whereinthe antibody is labeled and used for tumor imaging.

[0276] Methods of labeling include, but are not limited to, radioactivelabeling; enzymatic labeling, wherein the enzyme directly or indirectlyproduces a detectable product; and fluorescent labeling.

[0277] Further, this invention provides the above-described methods,wherein the antibody is radioactive.

[0278] In addition, this invention provides a pharmaceutical compositioncomprising one or more of the the above-described vectors and a carrier.

[0279] Pharmaceutically acceptable carriers are well known to thoseskilled in the art and include, but are not limited to, 0.01-0.1M andpreferably 0.05M phosphate buffer or 0.8% saline. Additionally, suchpharmaceutically acceptable carriers may be aqueous or non-aqueoussolutions, suspensions, and emulsions. Examples of non-aqueous solventsare propylene glycol, polyethylene glycol, vegetable oils such as oliveoil, and injectable organic esters such as ethyl oleate. Aqueouscarriers include water, alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media. Parenteral vehiclesinclude sodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers such as thosebased on Ringer's dextrose, and the like. Preservatives and otheradditives may also be present, such as, for example, antimicrobials,antioxidants, chelating agents, inert gases and the like.

[0280] This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

[0281] Experimental Details

[0282] Cancer is a progressive multigenic disorder characterized bydefined changes in the transformed phenotype that culminates inmetastatic disease. Determining the molecular basis of progressionshould lead to new opportunities for improved diagnostic and therapeuticmodalities. Through the use of subtraction hybridization, a geneassociated with transformation progression in virus and oncogenetransformed rat embryo cells, progression elevated gene-3 (PEG-3), hasbeen cloned. PEG-3 shares significant nucleotide and amino acid sequencehomology with the hamster growth arrest and DNA damage inducible genegadd34 and a homologous murine gene, MyD116, that is induced duringinduction of terminal differentiation by interleukin-6 in murine myeloidleukemia cells. PEG-3 expression is elevated in rodent cells displayinga progressed transformed phenotype and in rodent cells transformed byvarious oncogenes, including Ha-ras, v-src, mutant type 5 adenovirus(Ad5) and human papilloma virus-18. The PEG-3 gene is transcriptionallyactivated in rodent cells, as is gadd34 and MyD116, after treatment withDNA damaging agents, including methyl methanesulfonate and gammairradiation. In contrast, only PEG-3 is transcriptionally active inrodent cells displaying a progressed phenotype. Although transfection ofPEG-3 into normal and AdS-transformed cells only marginally suppressescolony formation, stable overexpression of PEG-3 in Ad5-transformed ratembryo cells elicits the progression phenotype. These results indicatethat PEG-3 is a new member of the gadd and MyD gene family with similaryet distinct properties and this gene may directly contribute to thetransformation progression phenotype. Moreover, these studies supportthe hypothesis that constitutive expression of a DNA damage response maymediate cancer progression.

[0283] First Series of Experiments

[0284] Materials and Methods

[0285] Cell Lines, Culture Conditions and Anchorage-Independent GrowthAssays. The isolation, properties and growth conditions of the E11,E11-NMT, E11-NMT×CREF somatic cell hybrids, E11×E11-NMT somatic cellhybrids and the E11-NMT AZA clones have been described (1,7-13). E11-rasR12 and E11-HPV E6/E7 clones were isolated by transfection with theHa-ras or the HPV-18 E6/E7 genes, respectively. The isolation,properties and growth conditions of CREF, CREF-H5hr1 A2, CREF-ras, theCREF-ras/Krev1 B1, B1 T and B1 M and the CREF-ras/Krev1 B2, B2 T, and B2M clones have been described (21). CREF-src and CREF-HPV 18 clones wereisolated by transfection with the v-src and HPV-18 E6/E7 genes,respectively. All cells were grown in Dulbecco's modified Eagle'sminimum essential medium supplemented with 5% fetal bovine serum at 37°C. in a 5% CO₂ plus 95% air humidified incubator. Anchorage independenceassays were performed by seeding various cell densities in 0.4% Nobleagar on a 0.8% agar base layer both of which contain growth medium (7).

[0286] Cloning and Sequencing of the PEG-3 cDNA. The PEG-3 gene wascloned from E11-NMT cells using subtraction hybridization as described(23). A full-length PEG-3 cDNA was obtained using the rapidamplification of cDNA end (RACE) procedure and direct ligation (25,26).Sequencing was performed by the dideoxy-chain termination (Sanger)method (27). The coding region of PEG-3 was cloned into a pZeoSV vector(Invitrogen) as described (25,26).

[0287] RNA Analysis and In Vitro Transcription Assays. Total cellularRNA was isolated by the guanidinium/phenol extraction method andNorthern blotting was performed as described (28). Fifteen μg of RNAwere denatured with glyoxal/DMSO and electrophoresed in 1% agarose gels,transferred to nylon membranes and hybridized sequentially with³²P-labeled PEG-3, Ad5 E1A and GAPDH probes (28,29). Followinghybridization, the filters were washed and exposed for autoradiography.The transcription rates of PEG-3, gadd34, MyD116, GAPDH and pBR322 wasdetermined by nuclear run-on assays (12,21).

[0288] In Vitro Translation of PEG-3. Plasmid, pZeoSV, containing PEG-3cDNA was linearized by digestion with Xho I and used as a template tosynthesize mRNA. In vitro translation of PEG-3 mRNA was performed with arabbit relticulocyte lysate translation kit as described by Promega.

[0289] DNA Transfection Assays. To study the effect of PEG-3 onmonolayer colony formation the vector (pZeoSV) containing no insert or apZeoSV-PEG-3 construct containing the PEG-3 coding region weretransfected into the various cell types by the lipofectin method(GIBCO/BRL) and Zeocin resistant clones were isolated or efficiency ofZeocin colony formation was determined (29,30).

[0290] Results and Discussion

[0291] Expression of the PEG-3 Gene Correlates Directly with theProgression Phenotype in Viral and Oncogene Transformed Rodent Cells. Acritical component of cancer development is progression, a process bywhich a tumor cell develops either qualitatively new properties ordisplays an increase in the expression of traits that enhance theaggressiveness of a tumor (1-4). Insight into this process offers thepotential of providing important new targets for intervening in theneoplastic process (1-4). In the Ad5 transformed RE cell culture modelsystem, enhanced anchorage-independent growth and in vivo tumorigenicaggressiveness, i.e., markers of the progression phenotype, are stabletraits that can be induced spontaneously or by gene transfer (oncogenesand growth factor-related genes) (Table 1). TABLE 1 Expression of PEG-3in Ad5-transformed RE cells directly correlates with expression of theprogression phenotype Agar Cloning Efficiency Tumorigenicity ProgressionCell Type^(a) (%)^(b) in Nude Mice^(c) Phenotype^(d) RE <0.001  0/10Prog⁻ CREF <0.001  0/18 Prog⁻ E11 2.9 ± 0.3 8/8 (36) Prog⁻ E11-NMT 34.3± 4.1  6/6 (20) Prog⁺ CREF × E11-NMT 2.0 ± 0.3 0/6 Prog⁻ F1 CREF ×E11-NMT 1.5 ± 0.1 0/6 Prog⁻ F2 CREF × E11-NMT 72.5 ± 9.4  3/3 (17) Prog⁺R1 CREF × E11-NMT 57.4 ± 6.9  3/3 (17) Prog⁺ R2 E11 × E11-NMT 5.6 ± 0.73/3 (56) Prog⁻ IIId E11 × E11-NMT 41.0 ± 4.9  3/3 (19) Prog⁺ IIIdTD E11× E11-NMT 0.3 ± 0.0 3/3 (44) Prog⁻ A6 E11 × E11-NMT 29.3 ± 3.5  N.T.Prog⁺ A6TD E11 × E11-NMT 1.5 ± 0.2 3/3 (31) Prog⁻ 3b E11 × E11-NMT 29.5± 2.8  3/3 (23) Prog⁺ IIA E11-NMT AZA C1 2.8 ± 0.5 N.T. Prog⁻ E11-NMTAZA B1 1.6 ± 0.3 3/3 (41) Prog⁻ E11-NMT AZA C2 2.0 ± 0.1 3/3 (50) Prog⁻E11-ras R12 36.8 ± 4.6  3/3 (18) Prog⁺ E11-HPV E6/E7 31.7 ± 3.1  3/3(22) Prog⁺ #N.T. = not tested.

[0292] Upon treatment of progressed cells with AZA, the progressionphenotype can be stably reversed (1,10). A reversion of progression alsooccurs following somatic cell hybridization of progressed cells withunprogressed Ads-transformed cells or with normal CREF cells. A furtherselection of these unprogressed Ad5-transformed cells by injection intonude mice results in acquisition of the progressed phenotype followingtumor formation and establishment in cell culture. These studiesdocument that progression in this model system is a reversible processthat can be stably produced by appropriate cellular manipulation. Inthis context, the Ad5-transformed RE model represents an importantexperimental tool for identifying genes that are associated with andthat mediate cancer progression.

[0293] To directly isolate genes elevated during progression we employedan efficient subtraction hybridization approach previously used to clonethe p21 gene (melanoma differentiation associated gene-6; mda-6) (23,25)and a novel cancer growth suppressing gene mda-7 (26,29). For thisapproach, cDNA libraries from a progressed mutant Ad5(H5ts125)-transformed RE clone, E11-NMT (10), and its parentalunprogressed cells, E11 (10,31), were directionally cloned into the λUni-ZAP phage vector and subtraction hybridization was performed betweendouble-stranded tester (E11-NMT) and single-stranded driver DNA (E11) bymass excision of the libraries (23). With this strategy in combinationwith the RACE procedure and DNA ligation techniques a full-length PEG-3cDNA displaying elevated expression in E11-NMT versus E11 cells wascloned. Northern blotting analysis indicates that PEG-3 expression is≧10-fold higher in all progressed Ad5-transformed RE cells, includingE11-NMT, specific E11-NMT×CREF somatic cell hybrid clones, R1 and R2,expressing an aggressive transformed phenotype and specific E11×E11-NMTsomatic cell hybrid clones, such as IIa that display the progressionphenotype (FIG. 1 and Table 1). PEG-3 mRNA levels also increasefollowing induction of progression by stable expression of the Ha-rasand HPV-18 E6/E7 oncogenes in E11 cells (FIG. 1). A further correlationbetween expression of PEG-3 and the progression phenotype is provided byE11×E11-NMT clones, such as IIId and A6, that initially display asuppression of the progression phenotype and low PEG-3 expression, butregain the progression phenotype and PEG-3 expression following tumorformation in nude mice, i.e., IIIdTD and A6TD (Table 1 and FIG. 1). Incontrast, unprogressed Ad5-transformed cells, including E11,E11-NMT×CREF clones F1 and F2, E11×E11-NMT clones IIId, A6 and 3b andAZA-treated E11-NMT clones B1, C1 and C2, have low levels of PEG-3 RNA.These results provide evidence for a direct relationship between theprogression phenotype and PEG-3 expression in this Ad5-transformed REcell culture system. They also demonstrate that the final cellularphenotype, i.e., enhanced anchorage-independence and aggressivetumorigenic properties, is a more important determinant of PEG-3expression than is the agent (oncogene) or circumstance (selection fortumor formation in nude mice) inducing progression.

[0294] A second rodent model used to study the process of cancerprogression employs CREF clones modified by transfection to expressdominant acting oncogenes (such as Ha-ras, v-src, HPV-18 and the mutantadenovirus H5hr1) and tumor suppressor genes (such as Krev-1, RB andwild-type p53) (19-22 and unpublished data). In this model system,Ha-ras-transformed CREF cells are morphologically transformed,anchorage-independent and induce both tumors and lung metastases insyngeneic rats and athymic nude mice (19-22). The Krev-1 (Ha-ras)suppressor gene reverses the in vitro and in vivo properties in Ha-rastransformed cells (21). Although suppression is stable in vitro,Ha-ras/Krev-1 CREF cells induce both tumors and metastases afterextended times in nude mice (21). Expression of PEG-3 is not apparent inCREF cells, whereas tumorigenic CREF cells transformed by v-src, HPV-18,H5hr1 and Ha-ras contain high levels of PEG-3 RNA (FIG. 2). Suppressionof Ha-ras induced transformation by Krev-1 inhibits PEG-3 expression.However, when Ha-ras/Krev-1 cells escape tumor suppression and formtumors and metastases in nude mice, PEG-3 expression reappears, withhigher expression in metastatic-derived than tumor-derived clones (FIG.2). These findings provide further documentation of a directrelationship between induction of a progressed and oncogenic phenotypein rodent cells and PEG-3 expression. As indicated above, it is thephenotype rather than the inducing agent that appears to be the primarydeterminant of PEG-3 expression in rodent cells.

[0295] The PEG-3 Gene Displays Sequence Homology with the Hamster gadd34and Mouse MyD116 Genes and is Inducible by DNA Damage. The cDNA sizes ofPEG-3, gadd34 and MyD116 are 2210, 2088 and 2275 nt, respectively. Thent sequence of PEG-3 is ˜73% and the aa sequence is ˜59% homologous tothe gadd34 (32) gene (FIG. 3 and data not shown). PEG-3 also sharessignificant sequence homology, ˜68% nt and ˜72% aa, with the murinehomologue of gadd34, MyD116 (33,34) (FIG. 3 and data not shown).Differences are apparent in the structure of the 3′ untranslated regionsof PEG-3 versus gadd34/MyD116. ATTT motifs have been associated withmRNA destabilization. In this context, the presence of 3 ATTT sequencesin Gadd34 and 6 tandem ATTT motifs in MyD116 would predict shorthalf-lives for these messages. In contrast, PEG-3 contains only 1 ATTTmotif suggesting that this mRNA may be more stable. The sequencehomologies between PEG-3 and gadd34/MyD116 are highest in the aminoterminal region of their encoded proteins, i.e., ˜69 and ˜76% homologywith gadd34 and Myd116, respectively, in the first 279 aa. In contrast,the sequence of the carboxyl terminus of PEG-3 significantly divergesfrom gadd34/Myd116, i.e., only ˜28 and ˜40% homology in the carboxylterminal 88 aa. In gadd34 and MyD116 a series of similar 39 aa arerepeated in the protein, including 3.5 repeats in gadd34 and 4.5 repeatsin MyD116. In contrast, PEG-3 contains only 1 of these 39 aa regions,with 64% and 85% homology to gadd34 and MyD116, respectively. On thebasis of sequence analysis, the PEG-3 gene should encode a protein of457 aa with a predicted MW of ˜50 kDa. To confirm this prediction, invitro translation analyses of proteins encoded by the PEG-3 cDNA weredetermined (FIG. 4). A predominant protein after in vitro translation ofPEG-3 has a molecular mass of ˜50 kDa (FIG. 4). In contrast, gadd34encodes a predicted protein of 589 aa with an M_(w) of ˜65 kDa andMyD116 encodes a predicted protein of 657 aa with an M_(w) of ˜72 kDa.The profound similarity in the structure of PEG-3 versus gadd34/MyD116cDNA and their encoded proteins suggest that PEG-3 is a new member ofthis gene family. Moreover, the alterations in the carboxyl terminus ofPEG-3 may provide a functional basis for the different properties ofthis gene versus gadd34/MyD116.

[0296] The specific role of the gadd34/MyD116 gene in cellularphysiology is not known. Like hamster gadd34 and its murine homologueMyD116, PEG-3 steady-state mRNA and RNA transcriptional levels areincreased following DNA damage by methyl methanesulfonate (MMS) andgamma irradiation (γIR) (FIGS. 2 and 5 and data not shown). In contrast,nuclear run-on assays indicate that only the PEG-3 gene istranscriptionally active (transcribed) as a function of transformationprogression (FIG. 5). This is apparent in CREF cells transformed byHa-ras and in E11-NMT and various E11-NMT subclones either expressing ornot expressing the progression phenotype (FIG. 5). The gadd34/MyD116gene, as well as the gadd45, MyD118 and gadd153 genes, encode acidicproteins with very similar and unusual charge characteristics (24).PEG-3 also encodes a putative protein with acidic properties similar tothe gadd and MyD genes (FIG. 3). The carboxyl-terminal domain of themurine MyD116 protein is homologous to the corresponding domain of theherpes simplex virus 1 γ₁34.5 protein, that prevents the prematureshutoff of total protein synthesis in infected human cells (35,36).Replacement of the carboxyl-terminal domain of γ₁34.5 with thehomologous region from MyD116 results in a restoration of function tothe herpes viral genome, i.e., prevention of early host shutoff ofprotein synthesis (36). Although further studies are required,preliminary results indicate that expression of a carboxyl terminusregion of MyD116 results in nuclear localization (36). Similarly,gadd45, gadd153 and MyD118 gene products are nuclear proteins (24,37).Moreover, both gadd45 and MyD118 interact with the DNA replication andrepair protein proliferating cell nuclear antigen (PCNA) and thecyclin-dependent kinase inhibitor p21 (37). MyD118 and gadd45 alsomodestly stimulate DNA repair in vitro (37). The carboxyl terminus ofPEG-3 is significantly different than that of MyD116 (FIG. 3). Moreover,the carboxyl-terminal domain region of homology between MyD116 and theγ₁34.5 protein is not present in PEG-3. In this context, thelocalization, protein interactions and properties of PEG-3 may bedistinct from gadd and MyD genes. Once antibodies with the appropriatespecificity are produced it will be possible to define PEG-3 locationwithin cells and identify potentially important protein interactionsmediating biological activity. This information will prove useful inelucidating the function of the PEG-3 gene in DNA damage response andcancer progression.

[0297] PEG-3 Lacks Potent Growth Suppressing Properties Characteristicof the gadd and Myd Genes. An attribute shared by the gadd and MyD genesis their ability to markedly suppress growth when expressed in human andmurine cells (24,37). When transiently expressed in various human tumorcell lines, gadd34/MyD116 is growth inhibitory and this gene cansynergize with gadd45 or gadd153 in suppressing cell growth (24). Theseresults and those discussed above suggest that gadd34/MyD116, gadd45,gadd153 and MyD118, represent a novel class of mammalian genes encodingacidic proteins that are regulated during DNA damage and stress andinvolved in controlling cell growth (24,37). In this context, PEG-3would appear to represent a paradox, since its expression is elevated incells displaying an in vivo proliferative advantage and a progressedtransformed and tumorigenic phenotype.

[0298] To determine the effect of PEG-3 on growth, E11 and E11-NMT cellswere transfected with the protein coding region of the PEG-3 gene clonedinto a Zeocin expression vector, pZeoSV (FIG. 6). This construct permitsan evaluation of growth in Zeocin in the presence and absence of PEG-3expression. E11 and E11-NMT cells were also transfected with the p21(mda-6) and mda-7 genes, previously shown to display growth inhibitoryproperties (25,26,29). Colony formation in both E11 and E11-NMT cells issuppressed 10 to 20%, whereas the relative colony formation followingp21 (mda-6) and mda-7 transfection is decreased by 40 to 58% (FIG. 6 anddata not shown). Colony formation is also reduced by 10 to 20% whenPEG-3 is transfected into CREF, normal human breast (HBL-100) and humanbreast carcinoma (MCF-7 and T47D) cell lines (data not shown). Althoughthe gadd and MyD genes were not tested for growth inhibition in E11 orE11-NMT cells, previous studies indicate colony formation reductionsof >50 to 75% in several cell types transfected with gadd34, gadd45,gadd153, MyD116 or MyD118 (24,37). The lack of dramatic growthsuppressing effects of PEG-3 and its direct association with theprogression state suggest that this gene may represent a unique memberof this acidic protein gene family that directly functions in regulatingprogression. This may occur by constitutively inducing signals thatwould normally only be generated during genomic stress. In this context,PEG-3 might function to alter genomic stability and facilitate tumorprogression. This hypothesis is amenable to experimental confirmation.

[0299] PEG-3 Induces a Progression Phenotype in Ad5-Transformed RECells. An important question is whether PEG-3 expression simplycorrelates with transformation progression or whether it can directlycontribute to this process. To distinguish between these twopossibilities we have determined the effect of stable elevatedexpression of PEG-3 on expression of the progression phenotype in E11cells. E11 cells were transfected with a Zeocin expression vector eithercontaining or lacking the PEG-3 gene and random colonies were isolatedand evaluated for anchorage independent growth (FIG. 7). A number ofclones were identified that display a 5- to 9-fold increase in agarcloning efficiency in comparison with E11 and E11-Zeocin vectortransformed clones. To confirm that this effect was indeed the result ofelevated PEG-3 expression, independent Zeocin resistant E11 cloneseither expressing or not expressing the progression phenotype wereanalyzed for PEG-3 mRNA expression (FIG. 8). This analysis indicatesthat elevated anchorage-independence in the E11 clones correlatesdirectly with increased PEG-3 expression. In contrast, no change in Ad5E1A or GAPDH mRNA expression is detected in the different clones. Thesefindings demonstrate that PEG-3 can directly induce a progressionphenotype without altering expression of the Ad5 E1A transforming gene.Further studies are required to define the precise mechanism by whichPEG-3 elicits this effect.

[0300] Cancer is a progressive disease characterized by the accumulationof genetic alterations in an evolving tumor (1-6). Recent studiesprovide compelling evidence that mutations in genes involved inmaintaining genomic stability, including DNA repair, mismatch repair,DNA replication, microsattelite stability and chromosomal segregation,may mediate the development of a mutator phenotype by cancer cells,predisposing them to further mutations resulting in tumor progression(38). Identification and characterization of genes that can directlymodify genomic stability and induce tumor progression will providesignificant insights into cancer development and evolution. Thisinformation would be of particular benefit in defining potentially noveltargets for intervening in the cancer process. Although the role ofPEG-3 in promoting the cancer phenotype remains to be defined, thecurrent studies suggest a potential causal link between constitutiveinduction of DNA damage response pathways, that may facilitate genomicinstability, and cancer progression. In this context, constitutiveexpression of PEG-3 in progressing tumors may directly induce genomicinstability or it may induce or amplify the expression of down-streamgenes involved in this process. Further studies are clearly warrantedand will help delineate the role of an important gene, PEG-3, in cancer.

[0301] Conclusion

[0302] Subtraction hybridization results in the identification andcloning of a gene PEG-3 with sequence homology and DNA damage inducibleproperties similar to gadd34 and MyD116. However, PEG-3 expression isuniquely elevated in all cases of rodent progression analyzed to date,including spontaneous and oncogene-mediated, and overexpression of PEG-3can induce a progression phenotype in Ad5-transformed cells. Our studiessuggest that PEG-3 may represent an important gene that is bothassociated with (diagnostic) and causally related to cancer progression.They also provide a potential link between constitutive expression of aDNA damage response pathway and progression of the transformedphenotype.

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[0317] 15. Fidler, I. J. (1990) Cancer Res. 50:6130-6138.

[0318] 16. Liotta, L. A., Steeg, P. G. & Stetler-Stevenson, W. G. (1991)Cell 64:327-336.

[0319] 17. Fidler, I. J. (1995) J. Natl. Cancer Inst. 87:1588-1592.

[0320] 18. Fisher, P. B., Babiss, L. E., Weinstein, I. B. & Ginsberg, H.S. (1982) Proc. Natl. Acad. Sci. USA 79: 3527-3531.

[0321] 19. Boylon, J. F., Jackson, J., Steiner, M., Shih, T. Y., Duigou,G. J., Roszman, T., Fisher, P. B. & Zimmer, S. G. (1990) Anticancer Res.10:717-724.

[0322] 20. Boylon, J. F., Shih, T. Y., Fisher, P. B. & Zimmer, S. G.(1992) Mol. Carcinog. 3:309-318.

[0323] 21. Su, Z.-z., Austin, V. N., Zimmer, S. G. & Fisher, P. B.(1993) Oncogene 8:309-318.

[0324] 22. Su, Z.-z., Yemul, S., Estabrook, A., Friedman, R. M., Zimmer,S. G. & Fisher, P. B. (1995) Intl. J. Oncology 7:1279-1284.

[0325] 23. Jiang, H. & Fisher, P. B. (1993) Mol. Cell. Different.1:285-299.

[0326] 24. Zhan, Q., Lord, K. A., Alamo, I., Jr., Hollander, M. C.,Carrier, F., Ron, D., Kohn, K. W., Hoffman, B., Liebermann, D. A. &Formace, A. J., Jr. (1994) Mol. Cell. Biol. 14:2361-2371.

[0327] 25. Jiang, H., Lin, J., Su, Z.-z., Kerbel, R. S., Herlyn, M.,Weissman, R. B., Welch, D. R. & Fisher, P. B. (1995) Oncogene10:1855-1864.

[0328] 26. Jiang, H., Lin, J. J., Su, Z.-z., Goldstein, N. I. & Fisher,P. B. (1995) Oncogene 11:2477-2486.

[0329] 27. Su, Z.-z., Leon, J. A., Jiang, H., Austin, V. A., Zimmer, S.G. & Fisher, P. B. (1993) Cancer Res. 53: 1929-1938.

[0330] 28. Jiang, H., Su, Z.-z., Datta, S., Guarini, L., Waxman, S. &Fisher, P. B. (1992) Intl. J. Oncol. 1:227-239.

[0331] 29. Jiang, H., Su, Z.-z., Lin, J. J., Goldstein, N. I., Young, C.S. H. & Fisher, P. B. (1996) Proc. Natl. Acad. Sci. USA 93:9160-9165.

[0332] 30. Su, Z.-z., Grunberger, D. & Fisher, P. B. (1991). Mol.Carcinog 4:231-242.

[0333] 31. Fisher, P. B., Weinstein, I. B., Eisenberg, D. & Ginsberg, H.S. (1978) Proc. Natl. Acad. Sci. USA 75:2311-2314.

[0334] 32. Formace, A. J., Jr., Alamo, I., Jr. & Hollander, M. C. (1988)Proc. Natl. Acad. Sci. USA 85:8800-8804.

[0335] 33. Lord, K. A., Hoffman-Liebermann, B. & Liebermann, D. A.(1990) Oncogene 5:387-396.

[0336] 34. Lord, K. A., Hoffman-Liebermann, B. & Liebermann, D. A.(1990) Nucleic Acids Res. 18:2823.

[0337] 35. Chou, J. & Roizman, B. (1994) Proc. Natl. Acad. Sci. USA91:5247-5251.

[0338] 36. He, B., Chou, J., Liebermann, D. A., Hoffman, B. & Roizman,B. (1996) J. Virol. 70:84-90.

[0339] 37. Vairapandi, M., Balliet, A. G., Formace, A. J., Jr., Hoffman,B. & Liebermann, D. A. (1996) Oncogene 11: 2579-2594.

[0340] 38. Loeb, L. A. (1994) Cancer Res. 54:5059-5063.

[0341] Second Series of Experiments

[0342] Development of Biosensor Systems to Efficiently and SelectivelyDetect Agents Inducing and Inhibiting DNA Damage Pathways, OncogenicTransformation and Cancer Progression

[0343] The PEG-3 gene is induced in a p53-independent manner in E11,CREF and human melanoma cells following treatment with DNA damagingagents, such as gamma irradiation (1 and unpublished data). Nuclearrun-on assays, that measure rates of gene transcription, indicate thatinduction of PEG-3 by DNA damage and expression of PEG-3 in cellsdisplaying the progression phenotype (such as E11-NMT and CREF cellstransformed by various oncogenes) involves elevated transcription ofthis gene (1). This data supports the hypothesis that the appropriatetranscriptional regulating factors are inducible following DNA damage incells and they are constitutively expressed in progressed cells. Sincetranscription of genes involves elements located in the promoter regionof genes, current data supports the hypothesis that the promoter regionof the PEG-3 gene is directly regulated as a function of genotoxicstress, oncogenic transformation and during cancer progression. Thisfinding will be exploited by isolating the promoter of PEG-3 (asdescribed below), linking this DNA sequence to a β-galactosidase (β-gal)reporter gene and constructing cells that either constitutively expressthis reporter gene (E11-NMT-β-gal, CREF-ras-β-gal and CREF-src-β-gal) orcells that contain a DNA damage inducible reporter gene (E11-β-gal andCREF-β-gal). The E11-NMT-β-gal, CREF-ras-β-gal and CREF-src-β-galconstructs can be used as sensitive and selective monitors for agentsthat can inhibit DNA damage and repair pathways, cancer progression andoncogene mediated transformation. Conversely, the E11-β-gal andCREF-β-gal cell constructs can be used as sensitive and selectivemonitors for conditions and agents that induce DNA damage and repairpathways and may also induce the progression and oncogene-mediatedtransformed phenotypes. The ability to modify β-gal expression, as afunction of activation or suppression of the PEG-3 promoter region orfactors that interact with the promoter region, can easily be assessedusing the appropriate substrate(5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside (X-gal) that isconverted into a final product (5-bromo-4-chloro-3-indole) that has ablue color. E11-NMT-β-gal cells will normally stain blue followingaddition of the appropriate substrate. However, should expression fromthe PEG-3 promoter region be suppressed this will extinguish β-galexpression thereby resulting in a loss of blue staining followingaddition of the substrate. These rapid, efficient and selectivebiosensor systems can easily be formatted for the screening of aninfinite number of compounds with potential cancer progressionsuppression, oncogene suppression and DNA damage inhibiting functions.E11-β-gal and CREF-β-gal cells will normally not stain blue followingaddition of the substrate. However, should the promoter region beactivated, following induction of appropriate DNA damage and repairpathways or expression of specific oncogenes, the β-gal gene will beactivated resulting in a blue stain following addition of the substrate.These rapid, efficient and selective biosensor systems can easily beformatted for the screening of an infinite number of compounds withpotential cancer progression, oncogene transformation and DNA damageinducing properties. These model systems will prove valuable inidentifying agents and elucidating pathways involved in cancerprogression, oncogenic transformation and DNA damage induction andrepair. This should lead to the development of novel therapeutics toprevent genomic damage and instability, thereby inhibiting cancerprogression and oncogene mediated-transformation, and the identificationof new classes of agents that can prevent DNA damage and enhance DNAdamage repair.

[0344] Identification and Characterization of the Promoter Region ofPEG-3, Cis-Acting Regulatory Elements of the PEG-3 Promoter andTrans-Acting Regulatory Elements That Activate (or Repress) PEG-3Expression.

[0345] Overview. Nuclear run-on studies indicate that the PEG-3 gene isconstitutively transcribed in progressed E11-NMT, CREF cells treatedwith methyl methanesulfonate (MMS) or gamma irradiation and inCREF-cells transformed by various oncogenes, such as Ha-ras and v-src.Studies will, therefore, be conducted to (i) clone the 5′-flankingregion of the PEG-3 gene and analyze its activity in E11 and E11-NMT,CREF and DNA damaged CREF and CREF cells transformed by variousoncogenes; (ii) identify cis-acting regulatory elements in the promoterregion of the PEG-3 gene which are responsible for the differentialinduction of expression in the different cell types and under differentexperimental conditions; and (iii) identify and characterizetrans-acting regulatory elements which activate (or repress) expressionof the PEG-3 gene.

[0346] To elucidate the mechanism underlying the transcriptionalregulation of the PEG-3 gene the 5′-flanking region of this gene will beanalyzed. This will be important for studies determining regulatorycontrol of the PEG-3 gene including autoregulation, developmentalregulation, tissue and cell type specific expression, DNA damageinduction and differential expression in cells displaying a progressedcancer phenotype. The isolation of the promoter region will also benecessary for creating a biosensor model for monitoring and analyzingfactors involved in mediating DNA damage and repair and oncogenictransformation and cancer progression. Once the appropriate sequence ofthe PEG-3 gene regulating the initiation of transcription has beenidentified, studies can be conducted to determine relevant trans-actingregulatory factors that bind to specific cis-acting regulatory elementsand activate or repress the expression of the PEG-3 gene. Thesemolecules may provide important clues for understanding the pathwaysgoverning DNA damage and repair mechanisms underlying cancerprogression. Ultimately, such an understanding may uncover importanttargets for directly modifying and intervening in these phenotypes andprocesses.

[0347] Cloning the promoter region of the PEG-3 gene and testing itsfunction. To identify the promoter region of PEG-3 we have used a humanPromoterFinder™ DNA Walking Kit (Clontech) (2,3), This PCR-based methodfacilitates the cloning of unknown genomic DNA sequences adjacent to aknown cDNA sequence. Using this approach an ˜2 kb fragment of PEG-3 thatmay contain the promoter region of this gene has been isolated. Theputative 5′ flanking-region of PEG-3 has been subcloned into thepBluescript vector and sequenced by the Sanger dideoxynucleotideprocedure. To verify the transcriptional start site deduced from thecDNA, primer extension analysis will be performed (4). In case of theidentification of multiple putative ATG or start sites RNase protectionassays will be performed using oligonucleotides spanning the 5′ end ofthe PEG-3 cDNA sequence (4,5). To define the boundary of the PEG-3promoter region, a heterologous expression system containing a bacterialchloramphenicol acetyltransferase (CAT) or luciferase gene withoutpromoter or enhancer will be employed (4,5,6). Putative promoter insertsof varying sizes will be subcloned into a CAT expression vector (6,7).Internal deletion constructs will be generated by taking advantage ofeither internal restriction sites or by partial digestion of internalsites if these sites are not unique. These constructs will betransfected into E11-NMT cells that display high levels of PEG-3expression. The CAT construct with minimal 5′-flanking region showingthe highest degree of expression will be identified as the PEG-3 genepromoter.

[0348] The functionality of the PEG-3 promoter will be determined inE11-NMT, CREF cells treated with MMS and gamma irradiation and CREFcells transformed by the Ha-ras and v-src oncogenes. Various CATconstructs will be transfected into these cell lines by the lipofectinmethod or electroporation (Gene Pulser, Bio-Rad) as previously described(4,8). To correct for DNA uptake and cell number used for eachtransfection experiment, the CAT constructs will be cotransfected withplasmids containing bacterial β-gal gene under the control of an Roussarcoma virus (RSV) promoter. The CAT and β-galactosidase enzymaticactivities will be determined using standard protocols (4,6,7). Minimal5′-flanking region displaying the highest CAT activity will beidentified as the promoter region for that tissue cell type orexperimental condition. If no induction of CAT activity is apparent,further subcloning and screening of cosmid or phage clones would beperformed until a PEG-3 promoter of sufficient length to mediate CATinduction in E11-NMT, CREF cells treated with MMS and gamma irradiationand CREF cells transformed by the Ha-ras and v-src oncogenes isobtained.

[0349] Once the promoter of PEG-3 is identified it will be subclonedinto a vector adjacent to a bacterial β-gal gene, PEG-3-Prom-β-galfusion (4). This construct will allow activation of the β-gal gene as afunction of transcription from the PEG-3 promoter. The vector constructwill also contain a bacterial antibiotic resistance gene, such as theneomycin or hygromycin gene, that will permit selection of cellscontaining the PEG-3-Prom-β-gal fusion. This vector will be transfectedinto E11, E11-NMT, CREF and CREF cells transformed by Ha-ras and v-srcand antibiotic resistant colonies will be selected in G418 (neomycingene) or hygromycin (hygromycin gene) as previously described (1,8,9).Antibiotic resistant colonies will be isolated and maintained asindependent cell lines. Clones constitutively expressing thePEG-3-Prom-β-gal gene (E11-NMT and CREF cells transformed by the Ha-rasand v-src oncogenes) will be identified by growth in the appropriatesubstrate resulting in a blue color. Similarly, clones containing aninducible PEG-3-Prom-β-gal gene (E11 and CREF cells) will be identifiedby treating cells with MMS or gamma irradiation, incubation in theappropriate substrate and identifying clones that develop a blue color.Clones displaying the appropriate properties will be furthercharacterized by Southern blotting (DNA organization) and Northernblotting (RNA expression). Clones with constitutive or inducible β-galexpression will then be tested for alteration in expression as afunction of culture conditions (low serum, high cell density, etc.),exposure to various DNA damaging agents, incubation in agents known tospecifically inhibit or enhance oncogene and progression phenotypes(such as caffeic acid phenethyl ester, phorbol ester tumor promoters,farnesyl transferase inhibitors, etc.), chemotherapeutic agents, viralinfection, etc. These studies will provide useful baseline informationas to the potential use of the specific constructs as biosensor monitorsfor agents capable of modifying cancer progression, oncogenictransformation and DNA damage and repair pathways.

[0350] Identifying cis-acting elements in the PEG-3 promoter responsiblefor expression in progressed cancer cells, oncogene transformed CREFcells and DNA damaged cells. Once a functional PEG-3 promoter has beenidentified studies will be conducted to locate cis-acting elementsresponsible for expression of PEG-3 in E11-NMT, oncogene transformedCREF (Ha-ras and v-src) and MMS treated CREF cells. To identifycis-acting DNA sequences, the DNA fragment displaying maximal promoterfunction in a transient transfection assay in E11-NMT, oncogenetransformed CREF and MMS treated CREF cells will be sequenced. Potentialregulatory elements will be defined by comparison to previouslycharacterized transcriptional motifs. The importance of these sequencesin regulating PEG-3 expression will be determined by introducing pointmutations in a specific cis element into the promoter region usingpreviously described site-specific mutagenesis techniques (4,9-12) orwith recently described PCR-based strategies, i.e., ExSite™ PCR-basedsite-directed mutagenesis kit and the Chameleon™ double-strandedsite-directed mutagenesis kit (Stratagene, CA). The mutated promoterconstructs will be cloned into CAT expression vectors and tested fortheir effects on the promoter function by transfecting into differentcell types displaying CAT activity. If increased detection sensitivityis required, the various promoter region mutants will be subcloned intoa luciferase reporter construct (7).

[0351] Identifying trans-acting nuclear proteins that mediatetranscriptional enhancing activity of the PEG-3 in progressed cancercells, oncogene transformed CREF cells and in DNA damaged CREF cells.The current view on regulation of eukaryotic gene expression suggeststhat trans-acting proteins bind to specific sites within cis-elements ofa promoter region resulting in transcriptional activation (13,14).Experiments will be performed to identify trans-acting factors (nuclearproteins) and determine where these factors interact with cis-regulatoryelements. To achieve this goal, two types of studies will be performed,one involving gel retardation (gel shift) assays (4,15-17) and thesecond involving DNase-I footprinting (methylation interference) assays(4).

[0352] Gel shift assays will be used to analyze the interactions betweencis-acting elements in the PEG-3 promoter and trans-acting factors inmediating transcriptional control (4,15-17). To begin to identify thetrans-acting factors, different non-labeled DNAs (including TATA, CAT,TRE, Sp-I binding site, NFκB, CREB, TRE, TBP, etc.) can be used ascompetitors in the gel shift assay to determine the relationship betweenthe trans-acting factors and other previously identified transcriptionalregulators. It is possible that the trans-acting factors regulatingtranscriptional control of the PEG-3 gene may be novel. To identifythese factors extracts will be purified from E11-NMT cells by two cyclesof heparin-Sepharose column chromatography, two cycles of DNA affinitychromatography and separation on SDS-polyacrylamide gels (18,19).Proteins displaying appropriate activity using gel shift assays will bedigested in situ with trypsin, the peptides separated by HPLC and thepeptides sequenced (20). Peptide sequences will be used to synthesizedegenerate primers and RT-PCR will be used to identify putative genesencoding the trans-acting factor. These partial sequences will be usedwith cDNA library screening approaches and the RACE procedure, ifnecessary, to identify full-length cDNAs encoding the trans-actingfactors (21-23). Once identified, the role of the trans-acting factorsin eliciting PEG-3 induction following DNA damage in CREF andconstitutive expression in E11-NMT, CREF-ras and CREF-src cells will bedetermined.

[0353] The functionality of positive and negative trans-acting factorswill be determined by transiently and stably expressing these genes inE11 and E11-NMT cells to determine effects on the progression phenotype,CREF and CREF-ras and CREF-src cells to determine effects on oncogenetransformation and in CREF and MMS treated CREF cells to determine theeffects of DNA damage on PEG-3 induction. Positive effects would beindicated if overexpressing a positive trans-acting factor facilitatesprogression, expression of the oncogenic phenotype and/or a DNA-damageinducible response, whereas overexpressing a negative trans-actingfactor inhibits progression, oncogene transformation and/or a DNA-damageinducible response.

[0354] Antisense approaches will be used to determine if blocking theexpression of positive or negative trans-acting factors can directlymodify progression, oncogenic transformation and/or DNA damage repairpathways. A direct effect of positive trans-acting factors in affectingcellular phenotype would be suggested if antisense inhibition of thepositive acting factor partially or completely inhibits the progressionand oncogene transformation phenotypes and/or DNA-damage and repairpathways. Conversely, a direct effect of negative trans-acting factorsin inhibiting expression of PEG-3 and progression, oncogenetransformation and/or DNA-damage and repair pathways would be suggestedif antisense inhibition of the negative factor facilitates PEG-3expression and the progression, oncogene transformation and/orDNA-damage inducible response pathways. Depending on the resultsobtained, cis-element knockouts could be used to further define the roleof these elements in regulating PEG-3 expression.

[0355] For DNase-I footprinting assays, nuclear extracts from E11,E11-NMT, CREF, CREF-ras, CREF-src and MMS treated CREF cells will beprepared and DNase-I footprinting assays will be performed as described(4,6). The differential protection between nuclear extracts from E11-NMTand E11 and MMS treated CREF, CREF-ras and CREF-src cells will providerelevant information concerning the involvement of trans-acting factorsin activation and the location of specific sequences in thecis-regulatory elements of the PEG-3 promoter mediating this activation.If differential protection is not detected using this approach, thesensitivity of the procedure can be improved by using different sizedDNA fragments from the PEG-3 promoter region or by using partiallypurified nuclear extracts (4,6).

[0356] The studies briefly described above will result in theidentification and cloning of the PEG-3 promoter region, theidentification of cis-acting regulatory elements in the PEG-3 promoterand the identification of trans-acting regulatory elements that activate(or repress) expression of the PEG-3 gene in unprogressed and progressedcancer cells, untransformed and oncogene transformed cells and undamagedand DNA damaged cells. Experiments will also determine if cellscontaining a PEG-3-Prom-β-gal fusion gene can be used as a biosensormonitoring system for the progression, oncogene transformation and DNAdamage and repair pathways. These reagents will be useful in definingthe mechanism underlying the differential expression of PEG-3 inprogressed and oncogene transformed cancer cells and during induction ofDNA damage and repair. This information should prove valuable indesigning approaches for selectively inhibiting PEG-3 expression, andtherefore potentially modifying cancer and DNA damage resulting fromtreatment with physical and chemical carcinogens.

[0357] Identifying a human homologue of the rat PEG-3 gene and definingthe genomic structure and the pattern of expression of the PEG-3 gene.Probing Northern blots containing total cytoplasmic RNA from humanmelanoma cells displaying different stages of cancer progression, i.e.,normal melanocytes, early radial growth phase (RGP) primary humanmelanoma, early and late vertical growth phase (VGP) primary humanmelanoma and metastatic human melanoma cells, indicate that PEG-3expression is highest in more advanced metastatic human melanoma (FIG.9). Treatment of human melanoma cells, containing a wild-type p53 or amutant p53 gene, with gamma irradiation results in enhanced PEG-3expression (FIG. 10). These results suggest that a human homologue ofrat PEG-3 is present in human melanoma cells and induction of this genecorrelates with cancer progression and DNA damage. Human genomic clonesof PEG-3 will be isolated by screening a human melanoma genomic lambdalibrary with sequences corresponding to the carboxyl terminus of PEG-3(that is significantly different from gadd34 and MyD116) and by PCRbased genomic DNA amplification procedures (4) The isolated positiveclones will be characterized by restriction mapping, and suitablerestriction fragments will be subcloned into the pBluescript vector(Strategene) (24). Exons will be identified by hybridization of thegenomic fragments of a panel of PEG-3 clones and subsequent comparisonof the genomic DNA sequences to that of the cDNA (25,26). A givenintron/exon boundary will be indicated when the sequence from thegenomic clones diverges from that of the cDNA. The size of each intronwill be estimated by restriction mapping (4,25,26). An alternativeapproach for identifying intron/exon junctions will use a set ofdifferent restriction endonucleases to digest the human genomic DNAs.Restriction fragments resulting from this digestion will be ligated withappropriate cDNA sequences and the other specific primer to the linkersequences. By using a panel of PEG-3 cDNA oligonucleotides as primers,PCR products will be generated, that contain most, if not all, unclonedgenomic DNA adjacent to PEG-3 exon sequence (25,26). The PCR productsobtained will be cloned and sequenced to deduce the intron/exonboundaries of the PEG-3 gene.

[0358] Having a human genomic clone of PEG-3 will permit a directdetermination of possible structural alterations and mutations in thePEG-3 gene (or its promoter) in human cancers. Tumor and normal tissuesamples will be collected in pairs from patients. Genomic DNAs will beextracted from these samples (4) and analyzed by Southern blotting withappropriate restriction enzymes for possible heterozygous deletions,homozygous deletions, insertions and/or rearrangements (27,28). Todetect point mutations, pairs of oligonucleotide primers for the exonswill be designed for single-strand conformation polymorphism (SSCP)analysis (27,28).

[0359] The studies briefly described above will delineate the structureof the human PEG-3 gene and identify structural changes in the PEG-3gene (or its promoter) in cancer versus normal tissue. A high frequencyof structural alterations and mutations, especially those that canpotentially alter the expression and functionality of the PEG-3 protein,in normal versus cancer tissue or in early versus late stage cancers,would suggest that these alterations in the PEG-3 gene may be involvedin initiation and/or progression of this cancer. Additionally,experiments to determine the state of methylation of the PEG-3 promoterregion should prove informative (29).

[0360] If specific mutations in PEG-3 (or its promoter) are found tocorrelate with cancer development and/or evolution, the effect of suchmutations on the in vitro and in vivo biological properties of cells canbe determined. Mutations will be introduced that alter the normal PEG-3gene to generate a progression specific PEG-3 gene product. To achievethese goals, the PEG-3 gene will be mutagenized at specific sites, usingthe ExSite™ PCR-based site-directed mutagenesis kit and the Chameleon™double-stranded site-directed mutagenesis kit (Stratagene, La Jolla,Calif.). We have documented experience in introducing mutations indefined regions of the adenovirus genome and characterizing thesegenetic changes (9-12). Once identified and characterized, mutantconstructs of the PEG-3 gene will be transfected into appropriate targetcells to determine the effects of specific mutations in PEG-3 oncellular phenotype.

REFERENCES FOR THE SECOND SERIES OF EXPERIMENTS

[0361] 1. Su Z-z, Shi Y & Fisher P B (1994) Proc Natl Acad Sci USA, insubmission.

[0362] 2. Siebert P, Chen S & Kellogg D (1995) CLONTECHniques, X (2)L:1-3.

[0363] 3. Siebert P, Chenchik A, Kellogg D E, Lukyanov K A & Lukyanov SA (1995) Nucleic Acids Res, 23: 1087-1088.

[0364] 4. Sambrook J, Fritsch E F & Maniatis T. In: Molecular Cloning: ALaboratory Manual, 2nd edition, Cold Spring Harbor Laboratories Press,Cold Spring Harbor, N.Y., 1989.

[0365] 5. Duigou G J, Su Z-z, Babiss L E, Driscoll B, Fung Y-KT & FisherP B. (1991) Oncogene 6:1813-1824.

[0366] 6. Shen R, Goswami S K, Mascareno E, Kumar A & Siddiqui MAQ.(1991) Mol Cell Biol 11: 1676-1685.

[0367] 7. Fisher A L, Ohsako S & Caudy M. (1996) Mol Cell Biol16:2670-2677.

[0368] 8. Jiang H, Lin J J, Su Z-z, Goldstein N I & Fisher P B (1995)Oncogene 11:2477-2486.

[0369] 9. Babiss L E, Fisher P B & Ginsberg H S. (1984) J Virol49:731-740.

[0370] 10. Babiss L E, Fisher P B & Ginsberg H S. (1984) J Virol 52:389-395.

[0371] 11. Herbst R S, Hermo H Jr, Fisher P B & Babiss L E. (1988) JVirol 62:4634-4643.

[0372] 12. Su Z-z, Shen R, Young C S H & Fisher P B. (1993) Mol Carcinog8:155-166.

[0373] 13. Maniatis T, Goodbourn S & Fischer A. (1987) Science236:1237-1244.

[0374] 14. Ptashne M. (1988) Nature 335:683-689.

[0375] 15. Su Z-z, Yemul S, Stein C A & Fisher P B. (1995) Oncogene10:2037-2049.

[0376] 16. Jiang H, Lin J, Young S-m, Goldstein N I, Waxman S, Davila V,Chellappan SP & Fisher P B. (1995) Oncogene 11:1179-1189.

[0377] 17. Su Z-z, Shen R, O'Brian C A & Fisher P B. (1994) Oncogene9:1123-1132.

[0378] 18. Kamat J P, Basu K, Satyamoorthy L, Showe L & Howe C C (1995)Mol Rep Dev 41:8-15.

[0379] 19. Basu A, Dong B, Krainer A R & Howe C C (1997) Mol Cell Biol17:677-686.

[0380] 20. Aebersold R H, Leavitt R A, Saavedra R A, Hood L E & Kent S BH (1987) Proc Natl Acad Sci USA 84:6970-6974.

[0381] 21. Jiang H, Lin J, Su Z-z, Kerbel R S, Herlyn M, Weissman R B,Welch D R & Fisher P B. (1995) Oncogene 10: 1855-1864.

[0382] 22. Jiang H, Lin J J, Su Z-z, Goldstein N I & Fisher P B (1995)Oncogene 11:2477-2486.

[0383] 23. Lin J J, Jiang H & Fisher P B (1996) Mol Cell Different4:317-333.

[0384] 24. Reddy P G, Su Z-z & Fisher P B Methods in Molecular Genetics,vol. 1, KW Adolph, Ed, Academic Press, Inc, Orlando, Fla., pp 68-102,1993.

[0385] 25. Hong F D, Huang H-S, To H, Young L-J H S, Oro A, Bookstein R,Lee EY-HP & Lee W H (1989) Proc Natl Acad Sci USA 86:5502-5506.

[0386] 26. Sun J, Rose J B & Bird P (1995) J Biol Chem 270:16089-16096.

[0387] 27. Puffenberger E G, Hosoda K, Washington S S, Nakao K, dewit D,Yanagisawa M and Charkravarti A. (1994) Cell 79:1257-1266.

[0388] 28. Washimi O, Nagatake M, Osada H, Ueda R, Koshikawa T, Seki T,Takahashi T and Takahashi T (1995) Cancer Res 55:514-517.

[0389] 29. Babiss L E, Zimmer S G & Fisher P B (1985) Science228:1099-1101.

[0390] Third Series of Experiments

[0391] Expression of PEG-3 in human melanoma cells. Studies were alsoperformed to evaluate PEG-3 expression in human melanoma cells and todetermine whether induction or increased expression occurs during DNAdamage. PEG-3 is expressed de novo in advanced stage tumorigenic andmetastatic human melanoma cell lines (MeWo, WM239, C8161, F0-1 andH0-1), whereas expression is reduced in immortalized normal humanmelanocyte (FM516-SV) and RGP (WM35) and early VGP (WM278) primarymelanomas (FIG. 9). Moreover, PEG-3 expression is enhanced followingexposure to gamma irradiation, but is not elevated following a similardose of MMS (100 mg/ml) inducing PEG-3 expression in CREF cells (FIG.10). Using a p53 mutant and p53 wild-type human melanoma cell lines, itis apparent that PEG-3 induction by gamma irradiation in human melanomacan occur by a wild-type p53 independent pathway (FIG. 10). Theseresults indicate that the PEG-3 response is not restricted to rodentcells treated with specific DNA damaging agents, but instead is a moregeneral response in mammalian cells. Furthermore, there appears to be adirect relationship between PEG-3 expression and human melanomaprogression.

[0392] Clarifying the role of PEG-3 in human cancer progression. Todefine the role of the PEG-3 gene in human cancer progression it will beessential to obtain a human homoloque of this gene. This will beachieved by low stringency hybridization screening of a human melanomacDNA library (1) and by PCR-based approaches using primers designed fromthe rat PEG-3 sequences that are highly homologous with gadd34 andMyD116 (4,5). Once a full-length PEG-3 (Hu) cDNA is obtained it will besequenced and in vitro translated to insure production of theappropriate sized protein (3-5). This gene can then be used to definepatterns of expression, by Northern blotting analysis, in normal, benignand metastatic human tumor cell lines and primary patient-derivedsamples (2-5). This survey will indicate the level of coordinateexpression between PEG-3 and human cancer progression. Clearly, if PEG-3is shown to be a regulator of the progression phenotype in humanmalignancies, a large number of interesting and important experimentscould be conducted to amplify on this observation. However, thesestudies would not be in the current scope of this grant because oflimited personnel and resources. The types of studies that could andshould be conducted include: (a) production of monoclonal antibodiesinteracting with PEG-3 (Hu) and evaluation of these reagents for cancerdiagnostic purposes; (b) cellular localization studies with PEG-3 (Hu)monoclonal antibodies to define potential targets for activity; (c)mapping the chromosomal location of PEG-3 (Hu) in the genome todetermine any association between previously identified regionsassociated with cancer; (d) identification and characterization of thegenomic structure of PEG-3 (Hu) and determining if alterations instructure correlate with cancer progression; (e) determine by nuclearrun-on and mRNA degradation assays if PEG-3 (Hu) expression iscontrolled at a transcriptional or postranscriptional level; (f)identification and characterization, if PEG-3 expression is regulatedtranscriptionally, of the promoter region of PEG-3 (Hu) to define themechanism of regulation of this gene in progressed cancer cells; (q) theidentification and characterization of cis-acting elements andtrans-regulating factors (nuclear proteins) regulating PEG-3 (Hu)expression; (h) defining the role of PEG-3 expression in vivo bycreating knockout mice and tissue specific knockout mice; and (i)determining, using transgenic mice and the tyrosinase promoter, the roleof overexpression of PEG-3 in normal melanocyte development. Thesestudies would provide important information about a potentially excitingand novel gene with direct relevance to human cancer progression.

REFERENCES FOR THE THIRD SERIES OF EXPERIMENTS

[0393] 1. Jiang, H. and P. Fisher Use of a sensitive and efficientsubtraction hybridization protocol for the identification of genesdifferentially regulated during the induction of differentiation inhuman melanoma cells. Mol Cell Different. 1: 285-299, 1993.

[0394] 2. Jiang, H., et al. The melanoma differentiation associated genemda-6, which encodes the cyclin-dependent kinase inhibitor p21 isdifferentially expressed during growth, differentiation and progressionin human melanoma cells. Oncogene 10: 1855-1864, 1995.

[0395] 3. Jiang, H., et al. Subtraction hybridization identifies a novelmelanoma differentiation associated gene, mda-7, modulated during humanmelanoma differentiation, growth and progression. Oncogene, 11:2477-2486, 1995.

[0396] 4. Shen, R., et al. Identification of the human prostaticcarcinoma oncogene PTI-1 by rapid expression cloning and differentialRNA display. PNAS, USA 92: 6778-6782, 1995.

[0397] 5. Su, Z-Z, et al. Surface-epitope masking and expression cloningidentifies the human prostate carcinoma tumor antigen gene PCTA-1 amember of the galectin gene family. PNAS, USA, 93:7252-7257, 1996.

[0398] Fourth Series of Experiments

[0399] The present invention is based, in part, on the identification ofcertain cDNA molecules that correspond to progression-associated mRNAmolecules. As used herein, a progression-associated mRNA is a mRNA whoseexpression correlates with tumor cell progression (i.e., the level ofRNA is at least 2-fold higher in progressing tumor cells). Aprogression-associated cDNA molecule comprises the sequence of aprogression-associated mRNA (and/or a complementary sequence).Similarly, a progression-associated protein or polypeptide comprises asequence encoded by a progression-associated mRNA, where the level ofprotein or polypeptide correlates with tumor cell progression (i.e., thelevel of protein is at least 2-fold higher in progressing tumor cells).Progression-associated sequences described herein are also called“progression elevated” genes (PEG).

[0400] Progression-Associated Polynucleotides. Any polynucleotide thatencodes a progression-associated polypeptide, or a portion or variantthereof as described herein, is encompassed by the present invention.Such polynucleotides may be single-stranded (coding or antisense) ordouble-stranded, and may be DNA (genomic, cDNA or synthetic) or RNAmolecules. Additional non-coding sequences may, but need not, be presentwithin a polynucleotide of the present invention, and a polynucleotidemay, but need not, be linked to other molecules and/or supportmaterials.

[0401] Progression-associated polynucleotides may be prepared using anyof a variety of techniques. For example, such a polynucleotide may beamplified from human genomic DNA, from tumor cDNA or from cDNA preparedfrom any of a variety of tumor-derived cell lines (typically cell linescharacterized by a progression phenotype), via polymerase chain reaction(PCR). For this approach, sequence-specific primers may be designedbased on the sequences provided herein, and may be purchased orsynthesized. An amplified portion may then be used to isolate a fulllength gene from a human genomic DNA library or from a tumor cDNAlibrary, using well known techniques, as described below. Alternatively,a full length gene can be constructed from multiple PCR fragments.

[0402] cDNA molecules encoding a native progression-associated protein,or a portion thereof, may also be prepared by screening a cDNA libraryprepared from mRNA of a cell that is in progression, such as E11-NMT orMCF-7 cells, as described herein. Such libraries may be commerciallyavailable, or may be prepared using standard techniques (see Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y., 1989, and references citedtherein). A library may be a cDNA expression library and may, but neednot, be subtracted using well known subtractive hybridizationtechniques.

[0403] There are many types of screens that may be employed, includingany of a variety of standard hybridization methods. For initial screens,conventional subtractive hybridization techniques may be used.

[0404] A progression-associated cDNA molecule may be sequenced usingwell known techniques employing such enzymes as Klenow fragment of DNApolymerase I, Sequenase® (US Biochemical Corp., Cleveland Ohio) Taqpolymerase (Perkin Elmer, Foster City Calif.), thermostable T7polymerase (Amersham, Chicago, Ill.) or combinations of recombinantpolymerases and proofreading exonucleases such as the ELONGASEAmplification System (Gibco BRL, Gaithersburg, Md.). An automatedsequencing system may be used, using instruments available fromcommercial suppliers such as Perkin Elmer and Pharmacia.

[0405] The sequence of a partial cDNA may be used to identify apolynucleotide sequence that encodes a full lengthprogression-associated protein using any of a variety of standardtechniques. Within such techniques, a library (cDNA or genomic) isscreened using one or more polynucleotide probes or primers suitable foramplification. Preferably, a library is size-selected to include largermolecules. Random primed libraries may also be preferred for identifying5′ and upstream regions of genes. Genomic libraries are preferred forobtaining introns and extending 5′ sequence.

[0406] For hybridization techniques, a partial sequence may be labeled(e.g., by nick-translation or end-labeling with ³²P) using well knowntechniques. A bacterial or bacteriophage library is then screened byhybridizing filters containing denatured bacterial colonies (or lawnscontaining phage plaques) with the labeled probe (see Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories,Cold Spring Harbor, N.Y., 1989). Hybridizing colonies or plaques areselected and expanded, and the DNA is isolated for further analysis.cDNA clones may be analyzed to determine the amount of additionalsequence by, for example, PCR using a primer from the partial sequenceand a primer from the vector. Restriction maps and partial sequenced maybe generated to identify one or more overlapping clones. The completesequence may then be determined using standard techniques, which mayinvolve generating a series of deletion clones. The resultingoverlapping sequences are then assembled into a single contiguoussequence. A full length cDNA molecule can be generated by ligatingsuitable fragments, using well known techniques.

[0407] Alternatively, there are numerous amplification techniques forobtaining a full length coding sequence from a partial cDNA sequence. Inan embodiment, amplification is performed via PCR. Any of a variety ofcommercially available kits may be used to perform the amplificationstep. Primers may be designed using, for example, software well known inthe art. Primers are preferably 22-30 nucleotides in length, have a GCcontent of at least 50% and anneal to the target sequence attemperatures of about 68° C. to 72° C. The amplified region may besequenced as described above, and overlapping sequences assembled into acontiguous sequence.

[0408] One such amplification technique is inverse PCR (see Triglia etal., Nucl. Acids Res. 16:8186, 1988), which uses restriction enzymes togenerate a fragment in the known region of the gene. The fragment isthen circularized by intramolecular ligation and used as a template forPCR with divergent primers derived from the known region. Within analternative approach, sequences adjacent to a partial sequence may beretrieved by amplification with a primer to a linker sequence and aprimer specific to a known region. The amplified sequences are typicallysubjected to a second round of amplification with the same linker primerand a second primer specific to the known region. A variation on thisprocedure, which employs two primers that initiate extension in oppositedirections from the known sequence, is described in WO 96/38591.Additional techniques include capture PCR (Lagerstrom et al., PCRMethods Applic. 1:111-19, 1991), walking PCR (Parker et al., Nucl.Acids. Res. 19:3055-60,1991) and rapid amplification of cDNA end (RACE)procedures (see Jiang et al., Oncogene 10:1855-1864, 1995; Jiang et al.,Oncogene 11:2477-2486, 1995). Other methods employing amplification mayalso be employed to obtain a full length cDNA sequence.

[0409] In certain instances, it is possible to obtain a full length cDNAsequence by analysis of sequences provided in an expressed sequence tag(EST) database, such as that available from GenBank. In an embodiment,searches for overlapping ESTs may be performed using well known programs(e.g., NCBI BLAST searches), and such ESTs may be used to generate acontiguous full length sequence

[0410] It will be appreciated by those of ordinary skill in the artthat, as a result of the degeneracy of the genetic code, there are manynucleotide sequences that encode a polypeptide as described herein. Someof these polynucleotides bear minimal homology to the nucleotidesequence of any native gene. Nonetheless, polynucleotides that vary dueto differences in codon usage are specifically contemplated by thepresent invention.

[0411] As noted above, antisense polynucleotides and portions of any ofthe above sequences are also contemplated by the present invention. Inan embodiment, such polynucleotides may be prepared by any method knownin the art, including chemical synthesis by, for example, solid phasephosphoramidite chemical synthesis. Alternatively, RNA molecules may begenerated by in vitro or in vivo transcription of DNA sequences encodinga progression-associated protein, or a portion thereof, provided thatthe DNA is incorporated into a vector downstream of a suitable RNApolymerase promoter (such as T3, T7 or SP6). Large amounts of RNA probemay be produced by incubating labeled nucleotides with a linearizedProgression Elevated Gene-3 fragment downstream of such a promoter inthe presence of the appropriate RNA polymerase. Certain portions of aPEG-3 polynucleotide may be used to prepare an encoded polypeptide, asdescribed herein. In addition, or alternatively, a portion may functionas a probe (e.g., for diagnostic purposes, such as to monitor or studythe progression of cancer), and may be labeled by a variety of reportergroups, such as radionuclides, fluorescent dyes and enzymes. Suchportions are preferably at least 10 nucleotides in length, morepreferably at least 12 nucleotides in length and still more preferablyat least 15 nucleotides in length. Within certain preferred embodiments,a portion for use as a probe comprises a sequence that is unique to aPEG-3 gene. A portion of a sequence complementary to a coding sequence(i.e., an antisense polynucleotide) may also be used as a probe or tomodulate gene expression. cDNA constructs that can be transcribed intoantisense RNA may also be introduced into cells of tissues to facilitatethe production of antisense RNA.

[0412] Any polynucleotide may be further modified to increase stabilityin vivo. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends; the use ofphosphorothioate or 2′ O-methyl rather than phosphodiesterase linkagesin the backbone; and/or the inclusion of nontraditional bases such asinosine, queosine and wybutosine, as well as acetyl-methyl-, thio- andother modified forms of adenine, cytidine, guanine, thymine and uridine.

[0413] Nucleotide sequences as described herein may be joined to avariety of other nucleotide sequences using established recombinant DNAtechniques. For example, a polynucleotide may be cloned into any of avariety of cloning vectors, including plasmids, phagemids, lambda phagederivatives and cosmids. Vectors of particular interest includeexpression vectors, replication vectors, probe generation vectors andsequencing vectors. In an embodiment, a vector will contain an origin ofreplication functional in at least one organism, convenient restrictionendonuclease sites and one or more selectable markers. Additionalinitial, terminal and/or intermediate DNA sequences that, for example,facilitate construction of readily expressed vectors may also bepresent. For example, regulatory elements required for expressioninclude promoter sequences to bind RNA polymerase and transcriptioninitiation sequences for ribosome binding. A bacterial expression vectormay include a promoter such as the lac promoter and for transcriptioninitiation the ShineDalgarno sequence and the start codon AUG.Similarly, a eukaryotic expression vector may include a heterologous orhomologous promoter for RNA polymerase II, a downstream polyadenylationsignal, the start codon AUG, and a termination codon for detachment ofthe ribosome. Such vectors may be obtained commercially or assembledfrom the sequences described by methods well-known in the art, forexample, the methods described above for constructing vectors. Otherelements that may be present in a vector will depend upon the desireduse, and will be apparent to those of ordinary skill in the art.

[0414] For example, insert and vector DNA can both be exposed to arestriction enzyme to create complementary ends on both molecules whichbase pair with each other and are then ligated together with DNA ligase.Alternatively, linkers can be ligated to the insert DNA which correspondto a restriction site in the vector DNA, which is then digested with therestriction enzyme which cuts at that site. Other means are alsoavailable and known to an ordinary skilled practitioner.

[0415] In one embodiment, a rat PEG-3 sequence is cloned in the EcoRIsite of a pZeoSV vector. The resulting plasmid, designated pPEG-3, wasdeposited on Mar. 6, 1997 with the American Type Culture Collection(ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A. under theprovisions of the Budapest Treaty for the International Recognition ofthe Deposit of Microorganism for the Purposes of Patent Procedure. Theplasmid, pPEG-3, was accorded ATCC Accession Number 97911.

[0416] Vectors as described herein may be transfected into a suitablehost cell, such as a mammalian cell, by methods well-known in the art.Such methods include calcium phosphate precipitation, electroporationand microinjection.

[0417] Progression-Associated Polypeptides. Polypeptides within thescope of the present invention comprise at least a portion of aprogression-associated protein or variant thereof, where the portion isimmunologically and/or biologically active. A polypeptide may furthercomprise additional sequences, which may or may not be derived from anative progression-associated protein. Such sequences may (but need not)possess immunogenic or antigenic properties and/or a biologicalactivity.

[0418] As used herein, immunologically active polypeptides include, butare not limited to, a polypeptide that is recognized (i.e., specificallybound) by a B-cell and/or T-cell surface antigen receptor. In anembodiment, immunological activity may be assessed using well knowntechniques, such as those summarized in Paul, Fundamental Immunology,3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Suchtechniques include screening polypeptides derived from the nativepolypeptide for the ability to react with antigen-specific antiseraand/or T-cell lines or clones, which may be prepared using well knowntechniques. An immunologically active portion of aprogression-associated protein reacts with such antisera and/or T-cellsat a level that is not substantially lower than the reactivity of thefull length polypeptide (e.g., in an ELISA and/or T-cell reactivityassay). In an embodiment, such screens may be performed using methodswell known to those of ordinary skill in the art, such as thosedescribed in Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988. B-cell and T-cell epitopes may also bepredicted via computer analysis.

[0419] Biologically active polypeptides include, but are not limited to,polypeptides that possesses one or more structural, regulatory and/orbiochemical functions of the native progression-associated protein. Forexample, a polypeptide may induce progression in cells at levelscomparable to the level of native protein. Appropriate assays designedto evaluate the activity may then be designed based on existing assaysknown in the art, and on the assays provided herein.

[0420] As noted above, polypeptides may comprise one or more portions ofa variant of an endogenous protein, where the portion is immunologicallyand/or biologically active (i.e., the portion exhibits one or moreantigenic, immunogenic and/or biological properties characteristic ofthe full length protein). Preferably, such a portion is at least asactive as the full length protein within one or more assays to detectsuch properties. A polypeptide variant as used herein includes, but isnot limited to, a polypeptide that differs from a native protein insubstitutions, insertions, deletions and/or amino acid modifications,such that the antigenic, immunogenic and/or biological properties of thenative protein are not substantially diminished. In an emboidment, avariant retains at least 80% sequence identity to a native sequence. Inanother emboidment, a variant retains at least 90% sequence identity toa native sequence. In another emboidment, a variant retains at least 95%sequence identity to a native sequence. Guidance in determining whichand how many amino acid residues may be substituted, inserted, deletedand/or modified without diminishing immunological and/or biologicalactivity may be found using any of a variety of computer programs knownin the art, such as DNAStar software. In an embodiment, properties of avariant may be evaluated by assaying the reactivity of the variant withantisera and/or T-cells as described above and/or evaluating abiological property characteristic of the native protein.

[0421] In an embodiment, a variant contains conservative substitutions.A conservative substitution comprises a substitution wherein an aminoacid is substituted for another amino acid that has similar properties,such that one skilled in the art of peptide chemistry would expect thesecondary structure and hydropathic nature of the polypeptide to besubstantially unchanged. In an embodiment, amino acid substitutions maybe made on the basis of similarity on polarity, charge, solubility,hydrophobicity, hydrophilicity and/or the amphipathic nature of theresidues. For example, negatively charged amino acids include asparticacid and glutamic acid; positively charged amino acids include lysineand arginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine and valine;glycine and alanine; asparagine and glutamine; and serine, threonine,phenylalanine and tyrosine. Other groups of amino acids that mayrepresent conservative changes include: (1) ala, pro, gly, glu, asp,gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala,phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also,or alternatively, contain nonconservative changes.

[0422] Variants within the scope of this invention also includepolypeptides in which the primary amino acid structure of a nativeprotein is modified by forming covalent or aggregative conjugates withother polypeptides or chemical moieties such as glycosyl groups, lipids,phosphate, acetyl groups and the like. Covalent derivatives may beprepared, for example, by linking particular functional groups to aminoacid side chains or at the N- or C-termini.

[0423] The present invention also includes polypeptides with or withoutassociated native-pattern glycosylation. Polypeptides expressed in yeastor mammalian expression systems may be similar to or slightly differentin molecular weight and glycosylation pattern than the native molecules,depending upon the expression system. Expression of DNA in bacteria suchas E. coli provides non-glycosylated molecules. In an embodiment,N-glycosylation sites of eukaryotic proteins are characterized by theamino acid triplet Asn-A_(l)-Z, where A_(l) is any amino acid exceptPro, and Z is Ser or Thr. Variants having inactivated N-glycosylationsites can be produced by techniques known to those of ordinary skill inthe art, such as oligonucleotide synthesis and ligation or site-specificmutagenesis techniques, and are within the scope of this invention.Alternatively, N-linked glycosylation sites can be added to apolypeptide.

[0424] As noted above, polypeptides may further comprise sequences thatare not related to an endogenous progression-associated protein. Forexample, an N-terminal signal (or leader) sequence may be present, whichco-translationally or post-translationally directs transfer of thepolypeptide from its site of synthesis to a site inside or outside ofthe cell membrane or wall (e.g., the yeast a-factor leader). Thepolypeptide may also comprise a linker or other sequence for ease ofsynthesis, purification or identification of the polypeptide (e.g.,poly-His or hemagglutinin), or to enhance binding of the polypeptide toa solid support. Fusion proteins capped with such peptides may also beresistant to intracellular degradation in E. coli. Protein fusionsencompassed by this invention further include, for example, polypeptidesconjugated to an immunoglobulin Fc region or a leucine zipper domain asdescribed, for example, in published PCT Application Wo 94/10308.Polypeptides comprising leucine zippers may, for example, be oligomeric,dimeric or trimeric. All of the above protein fusions may be prepared bychemical linkage or as fusion proteins, as described below.

[0425] Also included within the present invention are alleles of aprogression-associated protein. Alleles are alternative forms of anative protein resulting from one or more genetic mutations (which maybe amino acid deletions, additions and/or substitutions), resulting inan altered mRNA. Allelic proteins may differ in sequence, but overallstructure and function are substantially similar.

[0426] Progression-associated polypeptides, variants and portionsthereof may be prepared from nucleic acid encoding the desiredpolypeptide using well known techniques. To prepare an endogenousprotein, an isolated cDNA may be used. To prepare a variant polypeptide,standard mutagenesis techniques, such as oligonucleotide-directedsite-specific mutagenesis may be used, and sections of the DNA sequencemay be removed to permit preparation of truncated polypeptides. Briefly,host cells of a vector system containing a PEG-3 sequence under suitableconditions permitting production of the polypeptide may be grown, andthe polypeptide so produced may then be recovered.

[0427] Any of a variety of expression vectors known to those of ordinaryskill in the art may be employed to express recombinant polypeptides ofthis invention. Expression may be achieved in any appropriate host cellthat has been transformed or transfected with an expression vectorcontaining a DNA sequence that encodes a recombinant polypeptide.Suitable host cells include prokaryotes, yeast, insect cells and animalcells. In an embodiment, the host cells employed are E. coli, yeast,primary mammalian cells or a mammalian cell line such as COS, Vero,HeLa, fibroblast NIH3T3, CHO, Ltk⁻ or CV1. Following expression,supernatants from host/vector systems which secrete recombinant proteinor polypeptide into culture media may be first concentrated using acommercially available filter. Following concentration, the concentratemay be applied to a suitable purification matrix such as an affinitymatrix or an ion exchange resin. One or more reverse phase HPLC stepscan be employed to further purify a recombinant polypeptide.

[0428] Portions and other variants having fewer than about 100 aminoacids, and generally fewer than about 50 amino acids, may also begenerated by synthetic means, using techniques well known to those ofordinary skill in the art. For example, such polypeptides may besynthesized using any of the commercially available solid-phasetechniques, such as the Merrifield solid-phase synthesis method, whereamino acids are sequentially added to a growing amino acid chain. SeeMerrifield, J. Am. Chem. Soc. 85:2149-2146, 1963. Various modified solidphase techniques are also available (e.g., the method of Roberge et al.,Science 269:202-204, 1995). Equipment for automated synthesis ofpolypeptides is commercially available from suppliers such as AppliedBioSystems, Inc. (Foster City, Calif.), and may be operated according tothe manufacturer's instructions.

[0429] In an embodiment, an isolated polypeptide or polynucleotide isone that is removed from its original environment. For example, anaturally-occurring protein is isolated if it is separated from some orall of the coexisting materials in the natural system. A polynucleotideis considered to be isolated if, for example, it is cloned into a vectorthat is not a part of the natural environment.

[0430] Antibodies and Fragments Thereof. The present invention furtherprovides antibodies, and antigen-binding fragments thereof, thatspecifically bind to a progression-associated protein. In an embodiment,an antibody, or antigen-binding fragment specifically binds to aprogression-associated protein if it reacts at a detectable level(within, for example, an ELISA) with a progression-associated protein ora portion or variant thereof, and does not react detectably withunrelated proteins. In certain embodiments, antibodies that inhibitPEG-3 induced progression are used.

[0431] Antibodies may be prepared by any of a variety of techniquesknown to those of ordinary skill in the art. See, e.g., Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. Inan embodiment, antibodies can be produced by cell culture techniques,including the generation of monoclonal antibodies as described herein,or via transfection of antibody genes into suitable bacterial ormammalian cell hosts, in order to allow for the production ofrecombinant antibodies. In an embodiment, monoclonal antibodies may beproduced by in vitro techniques known to a person of ordinary skill inthe art.

[0432] Polypeptides comprising specific portions of a PEG-3 protein maybe selected for the generation of antibodies using methods well known inthe art. In general, hydrophilic regions are more immunogenic than thehydrophobic regions. In an embodiment, hydrophilic portions are used forthe generation of antibodies.

[0433] In one such technique, an immunogen comprising the polypeptide isinitially injected into any of a wide variety of mammals (e.g., mice,rats, rabbits, sheep or goats). In this step, the polypeptides of thisinvention may serve as the immunogen without modification.Alternatively, particularly for relatively short polypeptides, asuperior immune response may be elicited if the polypeptide is joined toa carrier protein, such as bovine serum albumin or keyhole limpethemocyanin. The immunogen is injected into the animal host, preferablyaccording to a predetermined schedule incorporating one or more boosterimmunizations, and the animals are bled periodically. Polyclonalantibodies specific for the polypeptide may then be purified from suchantisera by, for example, affinity chromatography using the polypeptidecoupled to a suitable solid support.

[0434] Monoclonal antibodies specific for the antigenic polypeptide ofinterest may be prepared, for example, using the technique of Kohler andMilstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto.Briefly, these methods involve the preparation of immortal cell linescapable of producing antibodies having the desired specificity (i.e.,reactivity with the polypeptide of interest). Such cell lines may beproduced, for example, from spleen cells obtained from an animalimmunized as described above. The spleen cells are then immortalized by,for example, fusion with a myeloma cell fusion partner, preferably onethat is syngeneic with the immunized animal. A variety of fusiontechniques may be employed. For example, the spleen cells and myelomacells may be combined with a nonionic detergent for a few minutes andthen plated at low density on a selective medium that supports thegrowth of hybrid cells, but not myeloma cells. In an embodiment, theselection technique uses HAT (hypoxanthine, aminopterin, thymidine)selection. After a sufficient time, usually about 1 to 2 weeks, coloniesof hybrids are observed. Single colonies are selected and their culturesupernatants tested for binding activity against the polypeptide.Hybridomas having high reactivity and specificity are preferred.

[0435] Monoclonal antibodies may be isolated from the supernatants ofgrowing hybridoma colonies. In addition, various techniques may beemployed to enhance the yield, such as injection of the hybridoma cellline into the peritoneal cavity of a suitable vertebrate host, such as amouse. Monoclonal antibodies may then be harvested from the ascitesfluid or the blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction. The antibodies of this invention may beused in the purification process in, for example, an affinitychromatography step. Antibodies with a high degree of specificity forPEG-3 may then be selected. Such antibodies may be used, for example, todetect the expression of PEG-3 in living animals, in humans, or inbiological tissues or fluids isolated from animals or humans.

[0436] In certain embodiments, antigen-binding fragments of antibodiesare used. Such fragments include Fab fragments, which may be preparedusing standard techniques. In an embodiment, immunoglobulins arepurified from rabbit serum by affinity chromatography on Protein A beadcolumns (Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, 1988) and digested by papain to yield Fab and Fcfragments. The Fab and Fc fragments may be separated by affinitychromatography on protein A bead columns.

[0437] Methods for Identifying Binding Agents and Modulating Agents. Thepresent invention further provides methods for identifying compoundsthat bind to and/or modulate the activity of a progression-associatedprotein. Such agents may be identified by contacting a polypeptide asprovided herein with a candidate compound or agent under conditions andfor a time sufficient to allow interaction with the polypeptide. Any ofa variety of well known binding assays may then be performed to assessthe ability of the candidate compound to bind to the polypeptide, andassays for a biological activity of the polypeptide may be performed toidentify agents that modulate (i.e., enhance or inhibit) the biologicalactivity of the protein. Depending on the design of the assay, apolypeptide may be free in solution, affixed to a solid support, presenton a cell surface or located intracellularly. Large scale screens may beperformed using automation.

[0438] Alternatively, compounds may be screened for the ability tomodulate expression (e.g., transcription) of PEG-3. For such assays apromoter for PEG-3 may be isolated using standard techniques. Thepresent invention provides nucleic acid molecules comprising such apromoter or a cis- or trans-acting regulatory element thereof. Suchregulatory elements may activate or suppress expression of PEG-3.

[0439] One method for identifying a promoter region uses a PCR-basedmethod to clone unknown genomic DNA sequences adjacent to a known cDNAsequence (e.g., a human PromoterFinder™DNA Walking Kit, available fromClontech). This approach may generate a 5′ flanking region, which may besubcloned and sequenced using standard methods. Primer extension and/orRNase protection analyses may be used to verify the transcriptionalstart site deduced from the cDNA.

[0440] To define the boundary of the promoter region, putative promoterinserts of varying sizes may be subcloned into a heterologous expressionsystem containing a suitable reporter gene without a promoter orenhancer may be employed. Suitable reporter genes may include genesencoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or the Green FluorescentProtein gene, and may be generated using well known techniques Internaldeletion constructs may be generated using unique internal restrictionsites or by partial digestion of non-unique restriction sites.Constructs may then be transfected into cells that display high levelsof PEG-3 expression (e.g., E11-NMT). In an embodiment, the constructwith the minimal 5′ flanking region showing the highest level ofexpression of reporter gene is identified as the PEG-3 gene promoter.

[0441] Once a functional PEG-3 promoter is identified, cis- andtrans-acting elements may be located. In an embodiment, cis-actingsequences may be identified based on homology to previouslycharacterized transcriptional motifs. Point mutations may then begenerated within the identified sequences to evaluate the regulatoryrole of such sequences. Such mutations may be generated usingsite-specific mutagenesis techniques or a PCR-based strategy. Thealtered promoter is then cloned into a reporter gene expression vector,as described above, and the effect of the mutation on reporter geneexpression is evaluated. Trans-acting factors that bind to cis-actingsequences may be identified using assays such as gel shift assays.Proteins displaying binding activity within such assays may be partiallydigested, and the resulting peptides separated and sequenced. Peptidesequences may be used to design degenerate primers for use within RT-PCRto identify cDNAs encoding the trans-acting factors.

[0442] To evaluate the effect of a candidate agent on PEG-3 expression,a promoter or regulatory element thereof may be operatively linked to areporter gene as described above. Such a construct may be transfectedinto a suitable host cell, such as E11-NMT or transfected forms of CREFTrans 6, including CREF-Trans 6:4NMT (expressing PTI-1), T24 (expressingras), CREF-src (expressing src) and CREF-HPV (expressing HPV). It hasbeen found, within the context of the present invention, that the PEG-3promoter is constitutively expressed in tumor cell lines, but not innormal cells. Clones that constitutively express high levels of reporterprotein may be selected and used within a variety of screens. Suchclones are encompassed by the present invention.

[0443] In an embodiment, cells may be used to screen a combinatorialsmall molecule library. Briefly, cells are incubated with the library(e.g., overnight). Cells are then lysed and the supernatant is analyzedfor reporter gene activity according to standard protocols. Compoundsthat result in a decrease in reporter gene activity are inhibitors ofPEG-3 transcription, and may be used to inhibit DNA damage and repairpathways, cancer progression and/or oncogene mediated transformation.

[0444] This invention further provides methods for identifying agentscapable of inducing DNA damage and repair pathways, cancer progressionand/or oncogene mediated transformation. Briefly, candidate compoundsmay be tested as described above, except that the cells employed (whichcomprise a PEG-3 promoter or regulatory element thereof operativelylinked to a reporter gene) are not in progression. For example,CREF-Trans 6 cells may be employed. Within such assays, an increase inexpression of the reporter gene after the contact indicates that thecompound is capable of inducing DNA damage and repair pathways, cancerprogression or oncogene mediated transformation.

[0445] Within other embodiments, cells may comprise one or moreexogenous suicidal genes under the control of a promoter or regulatoryelement of PEG-3. Such suicidal genes disrupt the normal progress of thecell following transcription from the promoter. Preferably, theswitching on of the suicidal gene will lead to cell death or halt incell growth. Example of such genes are genes which lead to apoptosis.

[0446] Pharmaceutical Compositions and Vaccines. Within certain aspects,compounds such as polypeptides, antibodies, nucleic acid moleculesand/or other agents that modulate PEG-3 expression or activity may beincorporated into pharmaceutical compositions or vaccines. In anembodiment, pharmaceutical compositions comprise one or more suchcompounds and a physiologically acceptable carrier. In an embodiment,certain vaccines may comprise one or more polypeptides and an immuneresponse enhancer, such as an adjuvant or a liposome (into which thecompound is incorporated). Pharmaceutical compositions and vaccines mayadditionally contain a delivery system, such as biodegradablemicrospheres which are disclosed, for example, in U.S. Pat. Nos.4,897,268 and 5,075,109. Pharmaceutical compositions and vaccines withinthe scope of the present invention may also contain other compounds,which may be biologically active or inactive.

[0447] A pharmaceutical composition or vaccine may contain DNA encodingan antisense polynucleotide or a polypeptides as described above, suchthat the polynucleotide or polypeptide is generated in situ. The DNA maybe present within any of a variety of delivery systems known to those ofordinary skill in the art, including nucleic acid expression systems,bacteria and viral expression systems. Appropriate nucleic acidexpression systems contain the necessary DNA sequences for expression inthe patient (such as a suitable promoter and terminating signal).Bacterial delivery systems involve the administration of a bacterium(such as Bacillus-Calmette-Guerrin) that expresses an immunogenicportion of the polypeptide on its cell surface. In a preferredembodiment, the DNA may be introduced using a viral expression system(e.g., vaccinia or other pox virus, retrovirus, or adenovirus), whichmay involve the use of a non-pathogenic (defective), replicationcompetent virus. Techniques for incorporating DNA into such expressionsystems are well known to those of ordinary skill in the art. The DNAmay also be “naked,” as described, for example, in Ulmer et al., Science259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993.The uptake of naked DNA may be increased by coating the DNA ontobiodegradable beads, which are efficiently transported into the cells.

[0448] While any suitable carrier known to those of ordinary skill inthe art may be employed in the pharmaceutical compositions of thisinvention, the type of carrier will vary depending on the mode ofadministration. Such carriers include, but are not limited to, aqueousor non-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents include propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, saline and buffered media.

[0449] Compositions of the present invention may be formulated for anyappropriate manner of administration, including for example, topical,oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous orintramuscular administration. Intravenous vehicles include fluid andnutrient replenishers, electrolyte replenishers such as those based onRinger's dextrose, and the like. For parenteral administration, such assubcutaneous injection, the carrier preferably comprises water, saline,alcohol, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's, a fixed oil, a fat, a wax or a buffer. For oraladministration, any of the above carriers or a solid carrier, such asmannitol, lactose, starch, magnesium stearate, sodium saccharine,talcum, cellulose, glucose, sucrose, and magnesium carbonate, may beemployed. Biodegradable microspheres (e.g., polylactate polyglycolate)may also be employed as carriers for the pharmaceutical compositions ofthis invention. For certain topical applications, formulation as a creamor lotion, using well known components, is preferred.

[0450] Such compositions may also comprise buffers (e.g., neutralbuffered saline or phosphate buffered saline), carbohydrates (e.g.,glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptidesor amino acids such as glycine, antioxidants, chelating agents such asEDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and/orpreservatives. Preservatives and other additives may also be present,such as, for example, antimicrobials, antioxidants, chelating agents,inert gases and the like. Compositions of the present invention may alsobe formulated as a lyophilizate. Compounds may also be encapsulatedwithin liposomes using well known technology.

[0451] Any of a variety of adjuvants may be employed in the vaccines ofthis invention to nonspecifically enhance the immune response. In anembodiment, the adjuvant contains a substance designed to protect theantigen from rapid catabolism, such as aluminum hydroxide or mineraloil, and a stimulator of immune responses, such as lipid A, Bortadellapertussis or Mycobacterium tuberculosis derived proteins. Suitableadjuvants are commercially available as, for example, Freund'sIncomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit,Mich.), Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.), alum,biodegradable microspheres, monophosphoryl lipid A and quil A.Cytokines, such as GM-CSF or interleukin-2, -7, or -12, may also be usedas adjuvants.

[0452] The compositions described herein may be administered as part ofa sustained release formulation (i.e., a formulation such as a capsuleor sponge that effects a slow release of compound followingadministration). In an embodiment, such formulations may be preparedusing well known technology and administered by, for example, oral,rectal or subcutaneous implantation, or by implantation at the desiredtarget site. Sustained-release formulations may contain a polypeptide,polynucleotide or antibody dispersed in a carrier matrix and/orcontained within a reservoir surrounded by a rate controlling membrane.Carriers for use within such formulations are biocompatible, and mayalso be biodegradable; preferably the formulation provides a relativelyconstant level of cyclic peptide release. The amount of active compoundcontained within a sustained release formulation depends upon the siteof implantation, the rate and expected duration of release and thenature of the condition to be treated or prevented.

[0453] Cancer Therapy. In further aspects of the present invention, thecompounds described herein may be used for therapy of cancer. Withinsuch aspects, the compounds (which may be polypeptides, antibodies,nucleic acid molecules or other modulating agents) are preferablyincorporated into pharmaceutical compositions or vaccines, asdescribed-above. Suitable patients for therapy may be any warm-bloodedanimal, preferably a human. A patient may or may not be afflicted withcancer, as determined by standard diagnostic methods. Accordingly, theabove pharmaceutical compositions and vaccines may be used to preventthe development of cancer or to treat a patient afflicted with cancer.

[0454] Within certain aspects, cells may be protected from therapeuticdamage (e.g., due to chemotherapy or a physical agent such asgamma-irradiation) and/or rendered resistant to progression byinhibiting or eliminating the expression and/or activity of PEG-3 in thecells. One method for inhibiting the expression of PEG-3 comprisesproviding an effective amount of antisense RNA in the cell. In anembodiment, such antisense technology can be used to control geneexpression through triple-helix formation, which compromises the abilityof the double helix to open sufficiently for the binding of polymerases,transcription factors or regulatory molecules (see Gee et al., In Huberand Carr, Molecular and Immunologic Approaches, Futura Publishing Co.(Mt. Kisco, N.Y.; 1994). Alternatively, an antisense molecule may bedesigned to hybridize with a control region of a gene (e.g., promoter,enhancer or transcription initiation site), and block transcription ofthe gene; or to block translation by inhibiting binding of a transcriptto ribosomes. In an embodiment, the expression of PEG-3 may beeliminated by deleting the gene or introducing mutation(s) into thegene.

[0455] Pharmaceutical compositions of the present invention may beadministered in a manner appropriate to the disease to be treated (orprevented). The route, duration and frequency of administration will bedetermined by such factors as the condition of the patient, the type andseverity of the patient's disease and the method of administration.Routes and frequency of administration may vary from individual toindividual, and may be readily established using standard techniques. Inan embodiment, the pharmaceutical compositions and vaccines may beadministered by injection (e.g., intracutaneous, intramuscular,intravenous or subcutaneous), intranasally (e.g., by aspiration) ororally. Preferably, between 1 and 10 doses may be administered over a 52week period. Preferably, 6 doses are administered, at intervals of 1month, and booster vaccinations may be given periodically thereafter.Alternate protocols may be appropriate for individual patients.

[0456] In an embodiment, an appropriate dosage and treatment regimenprovides the active compound(s) in an amount sufficient to providetherapeutic and/or prophylactic benefit. Such a benefit should resultsin an improved clinical outcome (e.g., more frequent remissions,complete or partial, or longer disease-free survival) in treatedpatients as compared to non-treated patients.

[0457] Appropriate dosages of polypeptides, polynucleotides, antibodiesand modulating agents may be determined using experimental models and/orclinical trials. In an embodiment, the use of the minimum dosage that issufficient to provide effective therapy is used. In an embodiment,patients may be monitored for therapeutic effectiveness using assayssuitable for the condition being treated or prevented, which will befamiliar to those of ordinary skill in the art. Suitable dose sizes willvary with the size of the patient, but will typically range from about0.1 mL to about 5 mL.

[0458] Cancer Detection, Diagnosis and Monitoring. Polypeptides,polynucleotides and antibodies, as described herein, may be used withina variety of methods for detecting a cancer, determining whether acancer is in progression, and monitoring the progression and/ortreatment of a cancer in a patient. Within such methods, any of avariety of methods may be used to detect PEG-3 activity or the level ofPEG-3 mRNA or protein in a sample. Suitable biological samples includetumor or normal tissue biopsy, mastectomy, blood, lymph node, serum orurine samples, or other tissue, homogenate or extract thereof obtainedfrom a patient.

[0459] Methods involving the use of an antibody may detect the presenceor absence of PEG-3 in any suitable biological sample. There are avariety of assay formats known to those of ordinary skill in the art forusing an antibody to detect polypeptide markers in a sample. See, e.g.,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988. For example, the assay may be performed in a Westernblot format, wherein a protein preparation from the biological sample issubmitted to gel electrophoresis, transferred to a suitable membrane andallowed to react with the antibody. The presence of the antibody on themembrane may then be detected using a suitable detection reagent, asdescribed below.

[0460] In another embodiment, the assay involves the use of antibodyimmobilized on a solid support to bind to the polypeptide and remove itfrom the remainder of the sample. The bound polypeptide may then bedetected using a second antibody or reagent that contains a reportergroup. Alternatively, a competitive assay may be utilized, in which apolypeptide is labeled with a reporter group and allowed to bind to theimmobilized antibody after incubation of the antibody with the sample.The extent to which components of the sample inhibit the binding of thelabeled polypeptide to the antibody is indicative of the reactivity ofthe sample with the immobilized antibody, and as a result, indicative ofthe concentration of polypeptide in the sample.

[0461] The solid support may be any material known to those of ordinaryskill in the art to which the antibody may be attached. For example, thesolid support may be a test well in a microtiter plate or anitrocellulose filter or other suitable membrane. Alternatively, thesupport may be a bead or disc, such as glass, fiberglass, latex or aplastic material such as polystyrene or polyvinylchloride. The supportmay also be a magnetic particle or a fiber optic sensor, such as thosedisclosed, for example, in U.S. Pat. No. 5,359,681.

[0462] The antibody may be immobilized on the solid support using avariety of techniques known to those in the art, which are amplydescribed in the patent and scientific literature. In the context of thepresent invention, immobilization includes both noncovalent association,such as adsorption, and covalent attachment (which may be a directlinkage between the antigen and functional groups on the support or maybe a linkage by way of a cross-linking agent). Immobilization byadsorption to a well in a microtiter plate or to a membrane ispreferred. In such cases, adsorption may be achieved by contacting theantibody, in a suitable buffer, with the solid support for a suitableamount of time. The contact time varies with temperature, but istypically between about 1 hour and 1 day. In an embodiment, contacting awell of a plastic microtiter plate (such as polystyrene orpolyvinylchloride) with an amount of antibody ranging from about 10 ngto about 1 μg, and preferably about 100-200 ng, is sufficient toimmobilize an adequate amount of polypeptide.

[0463] Covalent attachment of antibody to a solid support may also beachieved by first reacting the support with a bifunctional reagent thatwill react with both the support and a functional group, such as ahydroxyl or amino group, on the antibody. For example, the antibody maybe covalently attached to supports having an appropriate polymer coatingusing benzoquinone or by condensation of an aldehyde group on thesupport with an amine and an active hydrogen on the binding partnerusing well known techniques.

[0464] In certain embodiments, the assay for detection of polypeptide ina sample is a two-antibody sandwich assay. This assay may be performedby first contacting an antibody that has been immobilized on a solidsupport, commonly the well of a microtiter plate, with the biologicalsample, such that the polypeptide within the sample are allowed to bindto the immobilized antibody. Unbound sample is then removed from theimmobilized polypeptide-antibody complexes and a second antibody(containing a reporter group) capable of binding to a different site onthe polypeptide is added. The amount of second antibody that remainsbound to the solid support is then determined using a method appropriatefor the specific reporter group.

[0465] More specifically, once the antibody is immobilized on thesupport as described above, the remaining protein binding sites on thesupport are typically blocked. Any suitable blocking agent known tothose of ordinary skill in the art, such as bovine serum albumin orTween 20™ (Sigma Chemical Co., St. Louis, Mo.). The immobilized antibodyis then incubated with the sample, and polypeptide is allowed to bind tothe antibody. The sample may be diluted with a suitable diluent, such asphosphate-buffered saline (PBS) prior to incubation. In an embodiment,an appropriate contact time (i.e., incubation time) is that period oftime that is sufficient to detect the presence of polypeptide within asample obtained from an individual with cancer. Preferably, the contacttime is sufficient to achieve a level of binding that is at least 95% ofthat achieved at equilibrium between bound and unbound polypeptide.Those of ordinary skill in the art will recognize that the timenecessary to achieve equilibrium may be readily determined by assayingthe level of binding that occurs over a period of time. At roomtemperature, an incubation time of about 30 minutes is generallysufficient.

[0466] Unbound sample may then be removed by washing the solid supportwith an appropriate buffer, such as PBS containing 0.1% Tween 20™. Thesecond antibody, which contains a reporter group, may then be added tothe solid support. Preferred reporter groups include enzymes (such ashorseradish peroxidase), substrates, cofactors, inhibitors, dyes,radionuclides, luminescent groups, fluorescent groups and biotin. Theconjugation of antibody to reporter group may be achieved using standardmethods known to those of ordinary skill in the art.

[0467] The second antibody is then incubated with the immobilizedantibody-polypeptide complex for an amount of time sufficient to detectthe bound polypeptide. An appropriate amount of time may be determinedby assaying the level of binding that occurs over a period of time.Unbound second antibody is then removed and bound second antibody isdetected using the reporter group. The method employed for detecting thereporter group depends upon the nature of the reporter group. Forradioactive groups, scintillation counting or autoradiographic methodsare generally appropriate. Spectroscopic methods may be used to detectdyes, luminescent groups and fluorescent groups. Biotin may be detectedusing avidin, coupled to a different reporter group (commonly aradioactive or fluorescent group or an enzyme). Enzyme reporter groupsmay be detected by the addition of substrate (i.e. for a specific periodof time), followed by spectroscopic or other analysis of the reactionproducts.

[0468] To determine whether cells are in progression, expression ofPEG-3 in the cells is evaluated and compared with the level ofexpression in cells that are not in progression. In an embodiment, thesignal detected from the reporter group that remains bound to the solidsupport is compared to a signal that corresponds to a predeterminedcut-off value established from cells that are not in progression. In anembodiment, the cut-off value is the average mean signal obtained whenthe immobilized antibody is incubated with samples from cells that arenot in progression. In an embodiment, a sample generating a signal thatis three standard deviations above the predetermined cut-off value maybe considered positive for progression. In an embodiment, the cut-offvalue is determined using a Receiver Operator Curve, according to themethod of Sackett et al., Clinical Epidemiology: A Basic Science forClinical Medicine, p. 106-7 (Little Brown and Co., 1985). Briefly, inthis embodiment, the cut-off value may be determined from a plot ofpairs of true positive rates (i.e., sensitivity) and false positiverates (100%-specificity) that correspond to each possible cut-off valuefor the diagnostic test result. The cut-off value on the plot that isthe closest to the upper left-hand corner (i.e., the value that enclosesthe largest area) is the most accurate cut-off value, and a samplegenerating a signal that is higher than the cut-off value determined bythis method may be considered positive. Alternatively, the cut-off valuemay be shifted to the left along the plot, to minimize the falsepositive rate, or to the right, to minimize the false negative rate. Inan embodiment, a sample generating a signal that is higher than thecut-off value determined by this method is considered positive forprogression.

[0469] In a related embodiment, the assay is performed in a flow-throughor strip test format, wherein the antibody is immobilized on a membrane,such as nitrocellulose. In the flow-through test, the polypeptide withinthe sample bind to the immobilized antibody as the sample passes throughthe membrane. A second, labeled antibody then binds to theantibody-polypeptide complex as a solution containing the secondantibody flows through the membrane. The detection of bound secondantibody may then be performed as described above. In the strip testformat, one end of the membrane to which antibody is bound is immersedin a solution containing the sample. The sample migrates along themembrane through a region containing second antibody and to the area ofimmobilized antibody. Concentration of second antibody at the area ofimmobilized antibody indicates the presence of cells in progression.Typically, the concentration of second antibody at that site generates apattern, such as a line, that can be read visually. The absence of sucha pattern indicates a negative result. In an embodiment, the amount ofantibody immobilized on the membrane is selected to generate a visuallydiscernible pattern when the biological sample contains a level ofpolypeptide that would be sufficient to generate a positive signal inthe two-antibody sandwich assay, in the format discussed above.Preferably, the amount of antibody immobilized on the membrane rangesfrom about 25 ng to about 1 μg, and more preferably from about 50 ng toabout 1 μg. Such tests can typically be performed with a very smallamount of biological sample.

[0470] The presence or absence of cells in progression in a patient mayalso be determined by evaluating the level of mRNA encoding PEG-3 withinthe biological sample (e.g., a biopsy, mastectomy and/or blood samplefrom a patient) relative to a predetermined cut-off value. Such anevaluation may be achieved using any of a variety of methods known tothose of ordinary skill in the art such as, for example, in situhybridization and amplification by polymerase chain reaction. In anembodiment, probes and primers for use within such assays may bedesigned based on the sequences provided herein, or on similar sequencesidentified in other individuals. Probes may be used within well knownhybridization techniques, and may be labeled with a detection reagent tofacilitate detection of the probe. Such reagents include, but are notlimited to, radionuclides, fluorescent dyes and enzymes capable ofcatalyzing the formation of a detectable product.

[0471] Primers may be used within detection methods involving polymerasechain reaction (PCR), such as RT-PCR, in which PCR is applied inconjunction with reverse transcription. Typically, RNA is extracted froma sample tissue and is reverse transcribed to produce cDNA molecules.PCR amplification using specific primers generates aprogression-associated cDNA molecule, which may be separated andvisualized using, for example, gel electrophoresis. Amplification istypically performed on samples obtained from matched pairs of tissue(tumor and non-tumor tissue from the same individual) or from unmatchedpairs of tissue (tumor and non-tumor tissue from different individuals).The amplification reaction may be performed on several dilutions of cDNAspanning two orders of magnitude. A two-fold or greater increase inexpression in several dilutions of the tumor sample as compared to thesame dilutions of the non-tumor sample is typically considered positive.

[0472] Within certain specific embodiments, expression of PEG-3 may bedetected in a sample that contains cells by: (a) obtaining RNA from thecells; (b) contacting the RNA so obtained with a labeled (e.g.,radioactively) probe of PEG-3 under hybridizing conditions permittingspecific hybridization of the probe and the RNA; and (c) determining thepresence of RNA hybridized to the molecule. As noted above, mRNA may beisolated and hybridized using any of a variety of procedures well-knownto a person of ordinary skill in the art. The presence of mRNAhybridized to the probe may be determined by gel electrophoresis orother methods known in the art. By measuring the amount of the hybridformed, the expression of the PEG-3 protein by the cell can bedetermined. Alternatively, RNA obtained from the cells may be amplifiedby polymerase chain reaction (PCR) with appropriate primers derived froma known PEG-3 sequence. The presence of specific amplified DNA followingPCR is an indicative of PEG-3 expression in the cells.

[0473] Certain in vivo diagnostic assays may be performed directly onthe tumor. One such assay involves contacting tumor cells with anantibody or fragment thereof that binds to a progression-associatedprotein. The bound antibody or fragment may then be detected directly orindirectly via a reporter group. Such antibodies may also be used inhistological applications.

[0474] Within related aspects, the present invention provides methodsfor diagnosing the aggressiveness of cancer cells. Such methods areperformed as described above, wherein an increase in the amount of theexpression indicates that a cancer cell is more aggressive.

[0475] In other aspects of the present invention, the progression and/orresponse to treatment of a cancer may be monitored by performing any ofthe above assays over a period of time, and evaluating the change in thelevel of the response (i.e., the amount of polypeptide or mRNAdetected). For example, the assays may be performed every month to everyother month for a period of 1 to 2 years.

[0476] In an embodiment, a cancer is progressing in those patients inwhom the level of the response increases over time. In contrast, acancer is not progressing when the signal detected either remainsconstant or decreases with time.

[0477] The present invention further provides kits for use within any ofthe above diagnostic methods. Such kits typically comprise two or morecomponents necessary for performing the assay. Such components may becompounds, reagents and/or containers or equipment. For example, onecontainer within a kit may contain a monoclonal antibody or fragmentthereof that specifically binds to a progression-associated polypeptide.Such antibodies or fragments may be provided attached to a supportmaterial, as described above. One or more additional containers mayenclose elements, such as reagents or buffers, to be used in the assay.Such kits may also contain a detection reagent (e.g., an antibody) thatcontains a reporter group suitable for direct or indirect detection ofantibody binding.

[0478] Transgenic Organisms. The present invention also providestransgenic nonhuman living organism expressing PEG-3 protein. In anembodiment, the living organism is animal.

[0479] One means available for producing a transgenic animal, with amouse as an example, is as follows: Female mice are mated, and theresulting fertilized eggs are dissected out of their oviducts. The eggsare stored in an appropriate medium. PEG-3 DNA or cDNA is purified froma vector by methods well-known in the art. Inducible promoters may befused with the coding region of the DNA to provide an experimental meansto regulate expression of the trans-gene. Alternatively or in addition,tissue specific regulatory elements may be fused with the coding regionto permit tissue-specific expression of the trans-gene. The DNA, in anappropriately buffered solution, is put into a microinjection needle(which may be made from capillary tubing using a pipes puller) and theegg to be injected is put in a depression slide. The needle is insertedinto the pronucleus of the egg, and the DNA solution is injected. Theinjected egg is then transferred into the oviduct of a pseudopregnantmouse (a mouse stimulated by the appropriate hormones to maintainpregnancy but which is not actually pregnant), where it proceeds to theuterus, implants, and develops to term. As noted above, microinjectionis not the only method for inserting DNA into the egg cell, and is usedhere only for exemplary purposes.

EXAMPLE

[0480] Identification of Human PEG-3. This Example illustrates theidentification of a human PEG-3 cDNA molecule.

[0481] Initially, PEG-3 gene expression was examined in various humantumor cell lines using a rat PEG-3 cDNA 3′-end fragment as a probe underlow stringency conditions. Hybridization was performed at 65° C.overnight in the following solution: 800 μl of 5M NaCl, 80 pl of 0.5MEDTA, 2 ml of 1M Na(PO₄) (pH 6.4), 10 ml 10% SDS, 23.12 ml H₂O, for atotal of 40 ml. Following hybridization, washing was performed in 1×SSC,0.1%SDS at room temperature for 15 minutes, and then twice at 65° C. for30 minutes. Overnight exposures indicated that the MCF-7 cell linehighly expresses a human PEG-3 homolog and MCF-7 was used to providemRNA resources for the establishment of a cDNA library.

[0482] To establish an MCF-7 cDNA library, poly (A⁺) RNA was extractedand purified of from MCF-7 cells, and cDNA was generated using oligo(dT) as a primer through reverse transcription. λBk-MCV was used as avector to generate cDNA library. The original MCF-7 cDNA library wasgenerated with 1×10⁶ pfu and insert size was about 0.4 Kb-4 Kb.

[0483] The MCF-7 cDNA library was screened using a 600 bp rat PEG-3 cDNA3′-end fragment as a probe at low stringency. Prehybridization andhybridization were performed in the following solution: 100% Formamide 50 ml 20X SSC  25 ml 50X Denhardt's  10 ml 1 m Na(PO₄) (pH  5 ml 6.8)100 mg/ml SSDNA  1 ml 10% SDS  1 ml H₂O  8 ml 100 ml

[0484] Hybridization was performed at 42° C. overnight. Washing wasperformed in 1×SSC, 0.1% SDS at room temperature for 15 minutes, andthen twice at 65° C. for 30 minutes. Exposures were performed overnight.

[0485] Twenty-five positive clones were isolated from the MCF-7 cDNAlibrary using the above condition through primary screening, secondaryscreening, and third screening. After restriction mapping andsequencing, all 25 positive clones were confirmed to have an insert ofhuman PEG-3 cDNA 3′-end. The size for all these inserts was about400˜500 bp.

[0486] Northern blots were performed to evaluate the human PEG-3 geneexpression pattern in normal human tissues and human tumor cell lines,using a 400 bp human PEG-3 cDNA 3′-end fragment as a probe.

[0487] Hybridization was performed at 65° C. overnight in the followingsolution: 800 μl of 5M NaCl, 80 μl of 0.5M EDTA, 2 ml of 1M Na(PO₄) (pH6.4), 10 ml 10% SDS, 23.12 ml H₂O, for a total of 40 ml. Followinghybridization, washing was performed in 1×SSC, 0.1%SDS at roomtemperature for 15 minutes, and then twice at 65° C. for 30 minutes.Overnight exposures indicated 2 mRNA species of human PEG-3 gene thatexpress in a high level in most human tumor cell lines. These two mRNAspecies are about 1.5 and 2.8 Kb in size. No expression of PEG-3 genewas detected in all normal tissues except skeletal muscle whichexpresses 1.5 Kb species of human PEG-3 mRNAs in a low level.

[0488] 5′ RACE was used to generate the full length human PEG-3 cDNA,using poly (A⁺) RNA extracted from MCF-7 cells as a template. PEG genespecific primers were designed from the human PEG-3 cDNA 400 bpfragment, including primer A (CTAAGGCGTGTCCATGCTCTGGCC), primer B(CTCCTCTGCCTGGGCAATG) and primer C (CGAGCAAAGCGGCTTCGATC). First strandcDNA synthesis was carried out using human PEG-3 gene specific primer Aor B through reverse transcription. cDNA was purified by GlassMax DNAisolation spin cartridge purification and TdT tailed. PCR of dc-tailedcDNA was carried out using nested primer B or primer C. After PCR, thePCR products were separated using 1% agarose at 100 voltages for 1 hour.Two dominant fragments (1.7 Kb and 0.9 Kb) were observed afterelectrophoresis and cut for subcloning using AT cloning vector. Aftersequencing some subclones, the 1.7 Kb fragment was confirmed to coverall coding regions of the human PEG-3 cDNA, and 0.9 Kb fragment was atruncated product of the human PEG-3 cDNA with a start at the firstinternal repeat of PEG-3 cDNA and also had a 25 bp unique sequence atthe 5′-end. The 5′ and 3′ sequences of the 1.7 kb fragment are shown inFIG. 13.

[0489] The human PEG-3 gene also was found to express in human primarytumor samples using the RT-PCR method. Total RNAs extracted from primaryhuman tumor sample were used as template, with an oligo (dT) primer.Reverse transcription was carried out in 42° C. for one hour. For PCR,First strand cDNA generated from reverse transcription was used as thetemplate, and the primers were the human PEG-3 gene specific primersdesigned from the human PEG-3 gene cDNA 3′-end. PCR conditions were asfollows: Denaturation 94° C.-5′  1 cycle Denaturation 94° C.-30″Annealing of 60° C.-30′ primers {close oversize brace} 35 cycles Primerextension 72° C.-2′ Followed by Final extension 72° C.-7′ Indefinitehold  4° C., until samples are removed

[0490] Electrophoresis was used to separate PCR products of all testedsamples, in a 1.5% agarose gel, for 1 hour at 100 V.

[0491] Fifth Series of Experiments

[0492] Cancer is often a multistep process in which a tumor cell eitherdevelops qualitatively new phenotypes or an enhanced expression oftransformation related properties. Defining the molecular determinantsof progression should lead to improved cancer diagnosis and strategiesfor therapy. Subtraction hybridization identified a novel geneassociated with induction of transformation progression in virus andoncogene transformed rat embryo cells, progression elevated gene-3(PEG-3). PEG-3 expression correlates directly with the progressionphenotype in rodent cells. Ectopic expression of PEG-3 in transformedrodent cells elicits an aggressive oncogenic phenotype, whereasantisense inhibition of PEG-3 expression eliminates canceraggressiveness. PEG-3 has sequence homology to the growth arrest and DNAdamage inducible hamster gene gadd34, implicating DNA damage and repairprocesses in progression. A working hypothesis is that PEG-3 expressionis a downstream event in oncogenic transformation and progression andactivation of PEG-3 may directly alter the expression of genes involvedin cancer progression, including genes associated with tumorigenesis,metastasis and angiogenesis. Studies are evaluating the effect oftransient and stable expression of sense and antisense PEG-3 constructsin transformed cells on transformation progression in vitro and in vivo.Since induction of PEG-3 during progression and as a consequence of DNAdamage involves transcriptional activation, the promoter region of thePEG-3 gene has been isolated and will be investigated to identify andcharacterize cis-acting and trans-acting regulatory elements whichcontrol gene expression. Using the PEG-3 promoter, sensitive indicatorcell lines have been developed for identifying compounds capable ofinducing and inhibiting cancer progression. These studies are providingimportant insights into a novel progression gene with potentialrelevance to cancer development and evolution. The PEG-3 gene may serveas a target for selectively intervening in the progression process,thereby preventing cancer aggressiveness and metastasis.

[0493] Cancer is a progressive process with defined temporal stagesculminating in metastatic potential by evolving tumor cells. Althoughextensively scrutinized the molecular determinants of cancer progressionremain unclear. Well-characterized cell culture systems are valuableexperimental tools for defining the biochemical and molecular basis ofprogression. Two rodent model systems are providing insights into thegenes and processes regulating malignant progression of the transformedcell.

[0494] In adenovirus type 5 (Ad5) transformed rat embryo (RE) cells,progression can occur spontaneously by tumor formation in nude mice orby ectopic expression of oncogenes and signal transducinggrowth-regulating genes. In all contexts of progression, thedemethylating agent 5-azacytidine (AZA) can reverse this processresulting in an unprogressed phenotype in >95% of treated clones.Inhibition of progression also occurs in this system after formingsomatic cell hybrids between progressed and unprogressed cells. Using animmortal cloned rat embryo fibroblast (CREF) cell culture system,progression to metastasis and reversion of progression can be regulatedby appropriate genetic manipulation using the Ha-ras oncogene and theKrev-1 suppressor gene. These experimental findings support thehypothesis that progression may involve the selective inactivation ofgenes that suppress progression (progression suppressing genes) and/orthe induction of genes that promote progression (progression enhancinggenes). Identification and characterization of both types of geneticelements would prove of immense value for defining this importantcomponent of the cancer process and could provide useful targetmolecules for intervening in the neoplastic process.

[0495] To elucidate the molecular basis of progression we are using asubtraction hybridization approach. Subtraction hybridization betweenprogressed and unprogressed Ad5 transformed RE cells resulted in thecloning of progression elevated gene-3, PEG-3, that displays coordinateexpression with the progression and transformation phenotypes in Ad5 andoncogene transformed rat embryo cultures. PEG-3 is a novel gene sharingnucleotide (˜73 and ˜68%) and amino acid (˜59 and ˜72%) sequencehomology with the hamster growth arrest and DNA damage inducible genegadd34 and a homologous murine gene, MyD116, that is induced duringinduction of differentiation by IL6 in murine myeloid leukemia cells. Itis hypothesized that overexpression of PEG-3 in transformed andprogressed tumor cells may facilitate progression by regulating theexpression of genes that control the cancer process, including genesdirectly promoting tumorigenesis, metastasis and/or angiogenesis. Ourresearch is providing important information relative to the role of anovel DNA damage-inducible gene, PEG-3, in cancer development andprogression. This information should be valuable in designing refinedand sensitive techniques for cancer detection and for identifying cancerpreventing compounds. It may also provide a platform for developing newand improved cancer therapeutics.

[0496] The carcinogenic process involves a series of sequential changesin the phenotype of a cell resulting in the acquisition of newproperties or a further elaboration of transformation-associated traitsby the evolving tumor cell (1-4). Although extensively studied, theprecise genetic mechanisms underlying tumor cell progression during thedevelopment of most human cancers remain unknown. Possible factorscontributing to transformation progression, include: activation ofcellular genes that promote the cancer cell phenotype, i.e., oncogenes;activation of genes that regulate genomic stability, i.e., DNA repairgenes; activation of genes that mediate cancer aggressiveness andangiogenesis, i.e., progression elevated genes; loss or inactivation ofcellular genes that function as inhibitors of the cancer cell phenotype,i.e., tumor and progression suppressor genes; and/or combinations ofthese genetic changes in the same tumor cell (1-6). A useful model fordefining the genetic and biochemical changes mediating tumor progressionis the Ad5/early passage RE cell culture system (1,7-15). Transformationof secondary RE cells by AdS is often a sequential process resulting inthe acquisition of and further elaboration of specific phenotypes by thetransformed cell (7-10). Progression in the Ad5-transformation model ischaracterized by the development of enhanced anchorage-independence andtumorigenic capacity (as indicated by a reduced latency time for tumorformation in nude mice) by progressed cells (1,10). The progressionphenotype in Ad5-transformed rat embryo cells can be induced byselection for growth in agar or tumor formation in nude mice (7-10),referred to as spontaneous-progression, by transfection with oncogenes(11,14), such as Ha-ras, v-src, v-raf or E6/E7 region of human papillomavirus type-18 (HPV-18), referred to as oncogene-mediated progression, orby transfection with specific signal transducing genes (15), such asprotein kinase C (PKC), referred to as growth factor-related,gene-induced progression.

[0497] Progression, induced spontaneously or after gene transfer, is astable cellular trait that remains undiminished in Ad5-transformed REcells even after extensive passage (>100) in monolayer culture(1,10,14). However, a single-treatment with the demethylating agent AZAresults in a stable reversion in transformation progression in >95% ofcellular clones (1,10,11,14,15). The progression phenotype is alsosuppressed in somatic cell hybrids formed between normal or unprogressedtransformed cells and progressed cells (12-14). These findings suggestthat progression may result from the activation of specificprogression-promoting (progression elevated) genes or the selectiveinhibition of progression-suppressing genes, or possibly a combinationof both processes. To identify potential progression inducing genes withelevated expression in progressed versus unprogressed AdS-transformedcells we are using subtraction hybridization (14,16,17). The subtractionhybridization approach resulted in cloning of PEG-3 displaying elevatedexpression in progressed cells (spontaneous, oncogene-induced and growthfactor-related, gene-induced) than in unprogressed cells (parentalAd5-transformed, AZA-suppressed, and suppressed somatic cell hybrids)(17). These findings document a direct correlation between expression ofPEG-3 and the progression phenotype in this rat embryo model system.

[0498] The nucleotide sequence of PEG-3 is ˜73 and ˜68% and the aminoacid sequence is ˜59 and 72% homologous to gadd34 (18) and MyD116(19,20), respectively (17). The sequence homologies between PEG-3 andgadd34/MyD116 are highest in the amino terminal region of their encodedproteins, i.e., ˜69 and ˜76% homology with gadd34 and MyD116respectively, in the first 279 aa (17). In contrast, the sequence of thecarboxyl terminus of PEG-3 significantly diverges from gadd34/MyD116,i.e., only ˜28 and ˜49% homology in the carboxyl 88 aa (17). Thespecific function of the gadd34/MyD116 gene is not known. Like hamstergadd34 and its murine homologue MyD116, PEG-3 expression is induced inCREF cells by MMS and gamma irradiation (17). The gadd34/MyD116 gene, aswell as the gadd45, MyD118 and gadd153 genes, encode acidic proteinswith very similar and unusual charge characteristics (21). PEG-3 alsoencodes a putative protein with acidic properties similar to the gaddand MyD genes. The carboxyl-terminal domain of the murine MyD116 proteinis homologous to the corresponding domain of the herpes simplex virus 1γ₁34.5 protein, that prevents the premature shutoff of total proteinsynthesis in infected human cells (22,23). Replacement of thecarboxyl-terminal domain of γ₁34.5 with the homologous region fromMyD116 results in a restoration of function to the herpes viral genome,i.e., prevention of early host shutoff of protein synthesis (23).Although further studies are necessary, preliminary results indicatethat expression of a carboxyl terminus region of MyD116 results innuclear localization (23). Similarly, both gadd153 and gadd45 geneproducts are nuclear proteins (21). When transiently expressed invarious human tumor cell lines, gadd34/MyD116 is growth inhibitory andthis gene can synergize with gadd45 or gadd153 in suppressing cellgrowth (21). In contrast, ectopic expression of PEG-3 in normal CREF(cloned rat embryo fibroblast) and HBL-100 (normal breast epithelial)cells and cancer (E11 and E11-NMT (Ad5-transformed rat embryo) and MCF-7and T47D (human breast carcinoma) cells does not significantly inhibitcell growth or colony formation (17) (unpublished data). These resultssuggest that gadd34/MyD116, gadd45, gadd153 and MyD118, represent anovel class of mammalian genes encoding acidic proteins that areregulated during DNA damage and stress and involved in controlling cellgrowth. In this context, PEG-3 would appear to represent an enigma,since it is not growth suppressive and its expression is elevated incells displaying an in vivo proliferative advantage and a progressedtransformed and tumorigenic phenotype (17). PEG-3 may represent a uniquemember of this acidic protein gene family that directly functions inregulating progression, perhaps by constitutively inducing signals thatwould normally only be induced during genomic stress. Additionally,PEG-3 may modify the expression of down-stream genes involved inmediating cancer aggressiveness, i.e., tumor- and metastasis-mediatinggenes and genes involved in tumor angiogenesis. In these contexts, PEG-3could function to modify specific programs of gene expression and altergenomic stability, thereby facilitating tumor progression. Thishypothesis is amenable to experimental confirmation.

[0499] The final stage in tumor progression is the acquisition bytransformed cells of the ability to invade local tissue, survive in thecirculation and recolonize in a new area of the body, i.e., metastasis(24,25). Transfection of a Ha-ras oncogene into CREF cells (26) resultsin morphological transformation, anchorage-independence and acquisitionof tumorigenic and metastatic potential (27-29). Ha-ras-transformed CREFcells exhibit profound changes in the transcription and steady-statelevels of genes involved in suppression and induction of oncogenesis(29,30). Simultaneous overexpression of the Ha-ras suppressor geneKrev-1 in Ha-ras-transformed CREF cells results in morphologicalreversion, suppression of agar growth capacity and a delay in in vivooncogenesis (29). Reversion of transformation in Ha-ras+Krev-1transformed CREF cells correlates with a return in the transcriptionaland steady-state mRNA profile to that of nontransformed CREF cells(29,30). Following long latency times, Ha-ras+Krev-1 transformed CREFcells form both tumors and metastases in athymic nude mice (29). Thepatterns of gene expression changes observed during progression,progression suppression and escape from progression suppression supportsthe concept of transcriptional switching as a major component ofHa-ras-induced transformation (29,30).

[0500] Analysis of PEG-3 expression in CREF cells and variousoncogene-transformed and suppressor gene-reverted CREF cells indicates adirect relationship between PEG-3 expression and transformation andoncogenic progression (17). Northern blotting indicates that CREF cellsdo not express PEG-3, whereas PEG-3 expression occurs in CREF cellstransformed by several diverse-acting oncogenes, including Ha-ras,v-src, HPV 18 and mutant Ad5 (H5hr1) (17). Suppression of Ha-ras-inducedtransformation by Krev-1 results in suppression of PEG-3 expression.However, both tumor-derived and metastasis-derived Krev-1Ha-ras-transformed CREF cells express PEG-3. The highest relative levelsof PEG-3 mRNA are consistently found in the metastasis-derivedHa-ras+Krev-1 transformed CREF cells. These results indicate a directrelationship between PEG-3 expression and the transformed and oncogeniccapacity of CREF cells. In addition, PEG-3 expression directlycorrelates with human melanoma progression, with the highest levels ofexpression found in metastatic human melanoma and reduced levelsobserved in normal human melanocytes, radial growth phase (RGP) primarymelanomas and early vertical growth phase (VGP) primary melanomas(unpublished data). Although further studies with increased samples arerequired, these intriguing results suggest that PEG-3 may be relevant inhuman as well as rodent cancers.

[0501] A fundamentally important question is the role of PEG-3 in cancerprogression. PEG-3 could simply correlate with transformationprogression or alternatively it could directly regulate this process. Todistinguish between these possibilities, E11 cells (not expressingPEG-3) were genetically engineered to express PEG-3 (17). When assayedfor growth in agar or aggressiveness in vivo in nude mice, E11-PEG-3cells display a progression phenotype akin to that seen in E11-NMT cells(17.,31). Moreover, antisense inhibition of PEG-3 in E11-NMT (normallyexpressing PEG-3) results in suppression of the progression phenotype invitro and in vivo (31). Although the mechanism by which PEG-3 affectscancer progression in vivo remains to be determined, a potential rolefor induction of angiogenesis by PEG-3 is suggested (31). Tumorsisolated from nude mice infected with E11-NMT and E11-PEG-3 clones arehighly vascularized and they contain large numbers of blood vessels,whereas E11 and E11-NMT-PEG-3 AS tumors grow slower and they remaincompact without extensive blood vessel involvement (31). Further studiesare necessary to determine the mechanism by which PEG-3 expressionmodifies angiogenesis.

[0502] Experimental Studies

[0503] Model system for analyzing progression and suppression of thetransformed phenotype: oncogene-transformed and signal transductiongene-transformed early passage rat embryo (RE) cell cultures.Transformation of early passage RE cells with Ad5 or mutants of Ad5 is amultistep process involving temporal acquisition of enhancedtransformation-related phenotypes by the evolving transformed cells,i.e., progression. The progression process can be accelerated byselecting cells for increased anchorage-independence in vitro, injectingAd5-transformed cells into nude mice and isolating tumor-derived clonalcell lines or by transfection with specific oncogenes (including Ha-ras,V-src, V-raf and E6/E7 of HPV-18) or with signal transducing genes(including the β₁ isoform of PKC (7-15,16). In early passage RE cellstransformed by the mutant Ad5, H5ts125, the progression phenotype,induced spontaneously by nude mouse tumor formation or by transfectionwith the Ha-ras oncogene or the β₁ PKC gene, can be reversed by treatingcells with AZA (10,11,15). Suppression of the progression phenotype alsooccurs in intraspecific somatic cell hybrids formed between normal CREFand E11-NMT (spontaneously progressed nude-mouse tumor derivedH5ts125-transformed RE) cells or between E11 (non-progressedH5ts125-transformed RE) and E11-NMT cells (12,14). These findingsindicate that progression in Ad5-transformed, Ad5+oncogene (Ha-ras)transformed or Ad5+ signal transducing gene (β₁ PKC) transformed cellsis a reversible process that behaves genetically as a recessivephenotype. Progression may, therefore, involve the selectiveinactivation of progression-suppression genes and/or the activation ofprogression-inducing genes.

[0504] Identification and cloning genes associated with cancerprogression. To identify genes expressed at elevated levels inprogressed E11-NMT versus unprogressed E11 cells we are using asubtraction hybridization approach developed in our laboratory (16,17).For the subtraction hybridization approach, tester (E11-NMT) and driver(E11) cDNA libraries were directionally cloned into the commerciallyavailable λ Uni-ZAP phage vector and subtraction hybridization was thenperformed between double-stranded tester DNA (E11-NMT) andsingle-stranded driver DNA (E11) prepared by mass excision of thelibraries. The subtracted cDNAs were then cloned into the λ Uni-ZAPphage vector and used to probe Northern blots initially containingE11-NMT and E11 RNAs. cDNAs displaying elevated expression in E11-NMTversus E11 cells were identified, used to screen additional RNA samplesand appropriate clones were sequenced. One cDNA clone, PEG-3, displaysthe predicted association with expression of the progression phenotype(17). Expression of PEG-3 is apparent in a wide and diverse spectrum ofprogressed transformed RE clones, including spontaneously progressed(E11-NMT), progressed CREF×E11-NMT somatic cell hybrids (R1 and R2), aprogressed E11×E11-NMT somatic cell hybrid (IIa), progressedtumor-derived E11×E11-NMT somatic cell hybrids (A6-TD and IIId-TD), anHPV 18 progressed clone (E11-E6/E7), a Ha-ras progressed clone(E11-Ras-12) and a β₁ protein kinase C progressed clone (E11-PKC B1)(16). In contrast, PEG-3 expression is not detected or is apparent atreduced levels in the same series of cell lines that do not express theprogression phenotype, including unprogressed E11, unprogressedCREF×E11-NMT somatic cell hybrid clones (F1 and F2), unprogressedE11×E11-NMT somatic cell hybrid clones (IIId, A6 and 3b), andunprogressed E11-NMT subclones isolated after AZA treatment (E11-NMT-AZAclone C1, B1 and C2) (FIG. 15). These results document a directcorrelation between expression of progression and PEG-3 in RE cellsdisplaying specific stages of cancer progression (17).

[0505] Model system to analyze progression and suppression of thetransformed, tumorigenic and metastatic phenotype: Ha-ras-transformedand Ha-ras+Krev-1-transformed CREF cells. A second rodent model used tostudy the process of cancer progression employs a specific clone ofFischer rat embryo fibroblast cells, CREF, modified by transfection toexpress dominant acting oncogenes (such as Ha-ras, v-src, v-raf, and HPV18) and tumor suppressor genes (such as Krev-1, RB and p53)(27-30,32-34). In this model system, Ha-ras-transformed CREF cells aremorphologically transformed, anchorage-independent and induce bothtumors and lung metastases in syngeneic rats and athymic nude mice(27-30). The Krev-1 (Ha-ras) suppressor gene reverses the in vitro andin vivo properties in Ha-ras transformed cells (29,30). Althoughsuppression is stable in vitro, Ha-ras/Krev-1 CREF cells induce bothtumors and metastases after extended latency times in nude mice (29).CREF cells, as well as Ha-ras/Krev-1 reverted cells, contain RNAtranscripts and steady-state mRNA for several cancer suppressing genes,whereas these cells do not express transcripts or mRNAs for severalcancer promoting genes (29). During the processes of transformationsuppression and escape from transformation suppression changes in thetranscription and steady state RNA levels of defined genes are observed(29,30).

[0506] Expression of PEG-3 occurs in tumorigenic CREF cells transformedby v-src, HPV-18, H5hr1 (mutant of Ad5) and Ha-ras (17). Suppression ofHa-ras induced transformation by Krev-linhibits PEG-3 expression.However, when Ha-ras/Krev-1 cells escape tumor suppression and formtumors and metastases in nude mice, PEG-3 expression reappears.Treatment of CREF cells with gamma irradiation and MMS results in PEG-3expression by 4 hr and continued expression at 24 hr (17 and data notshown). These results indicate that PEG-3 expression is inducible by DNAdamage and suggests a direct association between PEG-3 expression andoncogenic transformation and tumor progression.

[0507] Analysis of PEG-3 in Rodent Progression Models.

[0508] (1) PEG-3 is a DNA damage-inducible gene. To define the level ofregulation of PEG-3 in normal and transformed cells nuclear run-onassays were performed (17). These studies document that PEG-3 istranscriptionally induced in CREF cells as a function of DNA damage,resulting from gamma irradiation or MMS treatment. The same DNA-damageinduction protocol also induces MyD116 and gadd34 transcription in CREFcells. In contrast, analysis of transformed CREF cells (Ha-ras),unprogressed rodent cells (E11, E11-NMT AZA C1 and E11×E11-NMT 3b) andprogressed rodent cells (E11-NMT and E11×E11-NMT IIa) indicate thatPEG-3, but not MyD116 or gadd34, is transcribed as a consequence oftransformation progression (17). These results document that PEG-3 is aDNA damage inducible gene that is constitutively expressed intransformed and progressed cells. They further demonstrate that aprimary level of regulation of PEG-3 occurs at a transcriptional level.

[0509] (2) PEG-3 lacks growth inhibitory and oncogenic transformationinducing properties. An attribute shared by the gadd and MyD genes istheir ability to markedly suppress growth when expressed in human andmurine cells (21,35). When transiently expressed in various human andmurine cell lines, gadd34/MyD116 is growth inhibitory and this gene cansynergize with gadd45 or gadd153 in suppressing cell growth (21). Todetermine the effect of PEG-3 on growth, E11 and E11-NMT cells weretransfected with the protein coding region of the PEG-3 gene cloned intoa Zeocin expression vector, pZeoSV (17). This construct permits anevaluation of growth in Zeocin in the presence and absence of PEG-3expression. E11 and E11-NMT cells were also transfected with the p21(mda-6) and mda-7 genes, previously shown to display growth inhibitoryproperties (36-38). Colony formation in both E11 and E11-NMT cells issuppressed 10-20% by PEG-3, whereas the relative colony formationfollowing p21 (mda-6) and mda-7 transfection is decreased by 40-58% (17and data not shown). Colony formation is also reduced by 10-20% whenPEG-3 is transfected into CREF, normal human breast (HBL-100), and humanbreast carcinoma (MCF-7 and T47D) cell lines (data not shown). Theseresults document that PEG-3 is distinct from the gadd and MyD genessince it does not significantly alter growth when expressed in varioushuman and rodent cell lines. To determine if PEG-3 has transformingability or if it can elicit an oncogenic phenotype in rodent cells,CREF-Trans 6 cells (39,40) were transfected with the PEG-3 gene in apZeoSV vector and analyzed for transformation in monolayer culture,growth in agar and tumor formation in athymic nude mice. PEG-3 did notinduce morphological transformation or growth in agar and pooled Zeocinresistant PEG-3 expressing CREF-Trans 6 cells did not produce tumors innude mice (data not shown). These results indicate that PEG-3 does nothave transforming or oncogenic potential when expressed in normal rodentcells.

[0510] (3) PEG-3 controls the progression phenotype in Ad5-transformedRE cells. A consequential question is whether PEG-3 expression simplycorrelates with transformation progression or whether it can directlycontribute or regulate this process. To distinguish between thesepossibilities we have determined the effect of stable elevatedexpression of PEG-3 on expression of the progression phenotype in E11cells. E11 cells were transfected with a Zeocin expression vector eithercontaining or lacking the PEG-3 gene, and random colonies were isolatedand evaluated for anchorage independent growth (17). A number of cloneswere identified that displayed a 5- to 9-fold increase in agar cloningefficiency in comparison with E11 and E11-Zeocin vector-transformedclones. Only the three PEG-3-transfected E11 clones displaying elevatedagar growth, i.e., E11-ZeoPEG-A, E11-ZeoPEG-B and E11-ZeoPEG-C,expressed PEG-3 mRNA (17). These findings demonstrate that PEG-3 candirectly induce a progression phenotype, as monitored by anchorageindependence, in H5ts125-transformed E11 cells.

[0511] (4) PEG-3 expression correlates with cancer aggressiveness andangiogenesis in Ad5-transformed RE cells. Studies were conducted todetermine the effect of forced PEG-3 expression in E11 cells and theconsequence of antisense inhibition of expression of PEG-3 in E11-NMTcells on tumorigenesis in nude mice. When injected subcutaneously intonude mice, stable PEG-3 expressing E11 induced tumors in 100% of animals(n=10) with a shorter latency time than observed with E11 and evenE11-NMT cells (data not shown). In contrast, E11-NMT cells containing anantisense PEG-3 gene display a reduction in agar colony formation (datanot shown) and an extension of tumor latency time in comparison withE11-NMT cells (data not shown). Tumors that developed were analyzed andfound to be significantly larger and highly vascularized in E11-PEG-3and E11-NMT cells as compared to E11 and E11-NMT AS PEG-3 cells (datanot shown). Sectioning of tumors indicate extensive blood vesselformation in E11-PEG-3 and E11-NMT cells but not in E11 parental cellsor AS PEG-3 expressing E11-NMT cells (data not shown). These resultsindicate that modifying PEG-3 expression in E11 and E11-NMT cells candirectly effect tumorigenesis and blood vessel formation (angiogenesis).

[0512] (5) Isolation and initial characterization of the PEG-3 promoter.To begin to define the mechanism by which DNA damage and progressiontranscriptionally induce PEG-3 expression we have identified a putativeregion of genomic DNA that contains the promoter of this gene. This wasachieved using the GenomeWalker™ Kit from CLONTECH (Palo Alto, Calif.)that relies on an approach described by Siebert et al. (41,42). Usingthis methodology a rat genomic DNA fragment upstream of the 5′untranslated region of the PEG-3 cDNA has been isolated and cloned. Thesize of this DNA fragment is ˜2.1 kb and its sequence is shown in FIG.14. This promoter has been linked to a luciferase reporter construct andevaluated for expression in different cell types. Additionally, PEG-Lucreporter constructs have been stably integrated into CREF-Trans 6, humanprostate cancer DNA transformed CREF-Trans 6 (CREF-Trans 6:4 NMT, 4NMT),Ha-ras-transformed CREF (CREF-Ha-ras), V-src-transformed CREF (CREF-src)and human papilloma virus 18-transformed CREF (CREF-HPV-18) cells. Inthese stable transfectants, luciferase is inducible by DNA damage(CREF-PEG-Luc) or constitutively expressed (4NMT-PEG-Luc,CREF-Ha-ras-Luc, CREF-src-Luc and CREF-HPV-18-Luc). Using a PEG-Lucreporter construct and transient transfection assays we demonstrateenhanced expression in progressed E11-NMT and E11-Ha-ras cells versusunprogressed E11 and E11-NMT-AZA cells (FIG. 15). In this system, thePEG-3 promoter is constitutively active in unprogressed cells and arelative increase of 5- to 10-fold is apparent in the progressed cells.Studies were also performed to determine if a relationship existsbetween oncogenic transformation induced by diverse oncogenes in CREFand CREF-Trans 6 cells and expression of the PEG-3 promoter (FIG. 16).In all cases of progression to an oncogenic phenotype the PEG-3 promoteris more active than in CREF or transformed CREF cells not displaying anoncogenic phenotype. The relative fold-induction of luciferase is higherin the CREF series than in the E11/E11-NMT series, whereas the absolutelevels of luciferase activity are lower in the CREF series. Thisreflects a lower de novo (essentially null) expression of PEG-3 in CREFand CREF-Trans 6 cells. The final test for promoter activity employedCREF-Trans 6 cells and CREF-Trans 6 cells containing a stablePEG-Luciferase gene (CREF-PEG-Luc cl 1). Using these cells wedemonstrate induction of luciferase activity in a temporal manner as afunction of DNA damage induced by treatment with MMS (100 μg/ml) (FIG.17). In the DNA damaged CREF cells the absolute levels of luciferasethat are induced are lower than found in oncogenically transformed CREFcells, thereby accounting for the lower relative fold increase apparentin luciferase activity. These results indicate that we have identifiedrat genomic sequences containing the promoter region of the PEG-3 genethat contains all of the elements necessary for responsiveness tocellular alterations occurring during cancer progression, oncogenictransformation and DNA damage.

[0513] Experimental Assay Protocols for Monitoring Luciferase Activity:Data presented in FIGS. 15 and 16. Cells were seeded at 2×10⁵/35-mmplate, ˜24 hr later cells were treated with lipofectin containing 4 μgof PEG-Luc plus a β-Gal control plasmid for 7 to 8 hr and the plateswere washed and incubated in complete medium for 48 hr. Cells were lysedin cell lysate buffer E3971 (Promega), added to luciferase substrateE1500 (Promega) and luciferase activity was determined using aluminometer. In FIG. 15, data reflects fold-change in luciferaseactivity versus E11 cells. In FIG. 16, data reflects fold-change inluciferase activity of transformed cells versus CREF cells. FIG. 17:CREF cells were treated as described above for 48 hr and then exposed to100 μg/ml of MMS for 24, 18, 8, 4 and 2 hr prior to lysate preparationand assaying for luciferase activity. Untransfected CREF-PEG-Luc(containing an integrated PEG-Luc gene) were treated with 100 μg/ml ofMMS for 24, 12, 8 and 4 hr prior to lysate preparation and assaying forluciferase activity. Data reflects fold-change in luciferase activityversus CREF or CREF-PEG-Luc, respectively. All luciferase activitieswere normalized to β-Gal activities.

[0514] Defining the mechanism underlying the differential expression ofPEG-3 as a function of cancer progression, oncogenic transformation andDNA damage. Nuclear run-on assays indicate that PEG-3 expressiondirectly correlates with an increase in the rate of RNA transcription(17). This association is supported by the isolation of a genomicfragment upstream of the 5′ untranslated region of the PEG-3 cDNA anddemonstration that this sequence linked to a luciferase reporter gene isactivated as a function of cancer progression, oncogenic transformationand DNA damage (FIGS. 15, 16 & 17). Additionally, changes in thestability of PEG-3 mRNA may also contribute to differential expressionof this gene as a function of cancer progression, oncogene expressionand DNA damage. To address this issue mRNA stability (RNA degradation)assays will be performed as described in detail previously (43). Ouranalysis focuses on the effect of cancer progression (E11-NMT, R1 and R2cells), oncogenic transformation (Ha-ras, V-src, H5hr1 and HPV-18transformed CREF cells) and DNA damage (gamma irradiation andMMS-treatment of CREF cells). Appropriate controls, E11, untransformedCREF cells and CREF cells not treated with DNA damaging agents,respectively, and experimental samples will be incubated withoutadditions or in the presence of 5 mg/ml of actinomycin D (in the dark),and 30, 60 and 120 min later, total cellular RNA will be isolated andanalyzed for gene expression using Northern hybridization. RNA blotswill be quantitated by densitometric analysis using a Molecular Dynamicsdensitometer (Sunnyvale, Calif.). These straight forward experimentswill indicate if the stability of PEG-3 is altered in cells as a directconsequence of spontaneous progression, expression of defined oncogenesor as a consequence of DNA damage.

[0515] Most eukaryotic genes are regulated at the level of initiation ofgene transcription. Detailed characterization of many differenteukaryotic transcriptional units has led to the general concept thatspecific interactions of short DNA sequences, usually located at the5′-flanking region of the corresponding genes (cis-acting elements),with certain cellular proteins (trans-acting elements) play a major rolein determining the rate of initiation of gene transcription. Toelucidate the mechanism underlying the transcriptional regulation of thePEG-3 gene the 5′-flanking region of this gene will be analyzed. Thiswill be important for determining regulatory control of the PEG-3 geneincluding autoregulation, developmental regulation, tissue and cell typespecific expression and differential expression in progressed versusunprogressed cells, enhanced expression as a function of oncogenictransformation and induction of expression as a consequence of DNAdamage. Once the appropriate regions of the PEG-3 gene regulating theinitiation of transcription has been confirmed, studies will beconducted to determine the relevant trans-acting regulatory factors thatbind to specific cis-acting regulatory elements and activate or repressexpression of the PEG-3 gene. The experiments outlined below aredesigned to: [1] define the 5′-flanking regions of the PEG-3 geneinvolved in mediating differential activity of PEG-3 in progressed,oncogenically transformed and DNA damaged cells; [2] identify cis-actingregulatory elements in the promoter region of the PEG-3 gene which areresponsible for the differential induction of PEG-3 expression; and [3]identify and characterize trans-acting regulatory elements that activate(or repress) expression of the PEG-3 gene.

[0516] (1) Primary analysis of the functional regions of the PEG-3promoter. Using a genomic walking strategy we have identified a5′-flanking promoter region of the PEG-3 gene that appears to encompassa functionally complete PEG-3 promoter (FIG. 14). To define importanttranscriptional regulatory regions of the PEG-3 promoter, a heterologousexpression system containing a luciferase gene without promoter orenhancer has been developed using the full-length promoter construct(44-46). Internal deletion mutations will be generated either by takingadvantage of internal restriction sites or by a nested exonuclease IIIbase deletion strategy. These constructs will be transfected into E11and E11-NMT, untransformed and transformed CREF (H5hr1, Ha-ras, v-srcand HPV-18) and control CREF and gamma irradiation or MMS treated CREFcells. On the basis of transfection analyses of various deletion andpoint mutations it will be possible to define elements responsible forinduction of PEG-3 as a consequence of cancer progression, specifictransformation pathways or DNA damage response.

[0517] Transcription of PEG-3 in E11-NMT cells, as determined by nuclearrun-on assays, is >20-fold higher than in E11 cells, whereas transienttransfection of the PEG-3 promoter-luciferase gene into these two celltypes indicates only an ˜5-fold increase in activity in E11-NMT versusE11 cells. This could indicate that the PEG-3 gene is repressed innon-expressing cells (such as E11) through a cis-acting mechanism thatis non-functional on transiently transfected promoters. Variousluciferase constructs will be transfected into the different cell typesby the lipofectamine method or electroporation (Gene Pulser, Bio-Rad) aspreviously described (44,47). To correct for DNA uptake and cell numberused for each transfection experiment, the luciferase constructs will betransfected with plasmids containing bacterial β-galactosidase geneunder the control of an Rous sarcoma virus (RSV) promoter (44-46).Studies will be conducted using multiple adult rat tissue Northern blots(CLONTECH) containing poly A⁺ RNA and probing with PEG-3 (as well asgadd34 and MyD116) to define which rat tissue normally express PEG-3.Previous studies document that genes expressing in more than one tissueoften require different sequences flanking the 5′-end of the gene. It ispossible that PEG-3 expression in any normal tissue or under differentcircumstances in rat cells, i.e., progression, oncogenic transformationor DNA damage, may be regulated by different 5′-sequences. In that case,we will obtain variable luciferase activities for different luciferaseconstructs in the various cell lines. Transcription motifs contributingto PEG-3 regulation in a tissue, cell type or specific progression,transformation or DNA damage pathway will thus be identified.

[0518] (2) Identifying cis-acting elements in the PEG-3 promoterresponsible for expression during cancer progression, oncogenictransformation and DNA damage. On the basis of the deletion studiesdescribed above, the potential location of cis-acting elementsresponsible for expression of PEG-3 during cancer progression, oncogenictransformation and DNA damage will be identified. The ˜2.1 kb PEG-3promoter has been sequenced and potential regulatory elements have beenidentified by comparison to previously characterized transcriptionalmotifs. The PEG-3 promoter contains a number of potentially importanttranscriptional motifs including PEA3 (AGGAAA), E2A (GCAGGTG), GRE(TGTTCT), E2F (TTTTGGCCG), TRE (GGTCA), acute phase reactive regulatingelement (GTGGGA), SP1 (GGGCGG), AP1 (TGACTCA), AP2 (TCCCCAACCC) and NF1(TGGATTTGAGCCA). The importance of these sequences in regulating PEG-3expression during cancer progression, oncogenic transformation and DNAdamage will be determined by introducing point mutations in a specificcis element into the promoter region using previously describedsite-specific mutagenesis techniques (44,47-50) or with recentlydescribed PCR-based strategies, i.e., ExSite™ PCR-based site-directedmutagenesis kit and the Chameleon™ double-stranded site-directedmutagenesis kit (Stratagene, CA). The mutated promoter constructs willbe cloned into luciferase expression vectors and tested for theireffects on the promoter function by transfection into different celltypes and monitoring luciferase activity. Since the promoter region forthe PEG-3 gene is located in front of the luciferase reporter gene inthe various pPEG-Luciferase constructs, the change in luciferaseactivity for each construct will permit a direct comparison of theactivity of the mutant promoter to that of the unmodified PEG-3promoter.

[0519] After the regulatory regions of the PEG-3 promoter are confirmedexperiments will be conducted to address a number of important questionsrelative to cancer progression, oncogenic transformation and DNA damageinduction of PEG-3 expression. (i) Nuclear run-on and transienttransfection assays with pPEG-Luciferase constructs will be used todetermine the effect of changes in DNA methylation (AZA and phenylbutyrate treatment) on PEG-3 expression in E11-NMT cells, treatment withdifferent classes of DNA damaging and cancer modulating agents (such asTPA, retinoids, UV-C, gamma irradiation, methylating carcinogens,topoisomerase inhibitors, okadaic acid, etc.) on PEG-3 expression inCREF and CREF-PEG-Luc cl 1 cells (PEG-Luciferase stably transformed CREFclone) and exposure to cancer modulating agents (such as the Krev-1gene, dominant negative inhibitors of specific oncogenes, chemicals suchas CAPE, retinoids, sodium butyrate, interferon, TNF-α and additionalprogression modulating agents) on PEG-3 expression in oncogenicallytransformed CREF cells (1,8-10,18,21,28,29,32,33,51); (ii) The level ofPEG-3 transcription in cells displaying different stages of cancerprogression and oncogenic transformation, including rodent model systemsof cancer progression (such as the Dunning rat prostate model,metastatic murine melanoma variants, etc.) and additional rodent cellstransformed by various oncogenes. These studies will indicate ifexpression of PEG-3 occurs in additional pathways of progression andtransformation. (iii) Transfection of varying lengths of the 5′ flankingregion and internal deletion luciferase constructs into rodent cellsdisplaying different stages of progression, transformed by differentclasses of oncogenes and treated with various DNA damaging and cancerpromoting and inhibiting agents. These regulatory elements will besequenced and compared with previously characterized transcriptionalmotifs to identify potential positive and negative regulatory elements;(iv) In addition to mutagenesis studies (to define functional motifsregulating transcriptional regulation of the PEG-3 promoter),cotransfection studies will be conducted with cDNAs containing putativepositive acting regulatory elements and a minimal PEG-3promoter-Luciferase construct into unprogressed and progressed rodentcells, untransformed CREF and oncogenically transformed CREF anduntreated and DNA damage treated CREF cells. These studies will indicateif the introduction of specific putative positive acting regulatoryelements can enhance PEG-3 expression in cells cotransfected with aminimal PEG-3 promoter region. The potential role of putative cis-actingnegative regulatory elements will be addressed by cotransfection with acomplete PEG-3 promoter region into the same target cells. These studieswill provide relevant information about the potential role of inhibitoryelements in regulating PEG-3 expression. (v) Experiments will also beperformed to evaluate the status of the endogenous PEG-3 gene duringcancer progression, oncogenic transformation and DNA damage. This willbe approached by using DNase hypersensitivity assays to look forstructural changes in this gene (44). Although not within the scope ofthe present studies, future studies could involve the identification ofa human PEG-3 cDNA, elucidation of the human PEG-3 promoter and analysisof the level of PEG-3 expression in human progression model systems.These studies would be quite informative in providing a potential linkbetween PEG-3 expression and cancer progression in human cells.

[0520] (3) Identifying trans-acting nuclear proteins that mediatetranscriptional enhancing activity of the PEG-3 gene during cancerprogression, oncogenic transformation and DNA damage. The current viewon regulation of eukaryotic gene expression suggests that trans-actingproteins bind to specific sites within cis-elements of a promoter regionresulting in transcriptional activation (52,53). Experiments will beperformed to identify trans-acting factors (nuclear proteins) anddetermine where these factors interact with cis-regulatory elements. Toachieve this goal, two types of studies will be performed, one involvinggel retardation (gel shift) assays (15,44,54,55) and the secondinvolving DNase-I footprinting (methylation interference) assays(44,56,57).

[0521] Gel shift assays will be used to analyze the interactions betweencis-acting elements in the PEG-3 promoter and trans-acting factors inmediating transcriptional control (15,54,55). For this assay,³²P-labeled cis-elements will be incubated with nuclear extracts fromE11 and E11-NMT, CREF and transformed CREF (Ha-ras, v-src, H5hr1 andHPV-18) and untreated CREF and CREF treated with MMS (100 μg/ml for 8hr) or gamma irradiation (10 Gy for 4 hr) and reaction mixtures will beresolved on 5 or 8% polyacrylamide gels. After autoradiography, thepattern of retarded DNAs on the gel will provide information concerningthe interaction between trans-acting factors and specific regions of thecis-acting elements in the PEG-3 promoter. Non-labeled cis-actingelements (self-competition) will be added as a competitor to duplicatesamples to eliminate the possibility of non-specific binding and toconfirm that the interaction is really conferred by the trans-actingfactor. To begin to identify the transacting factors, differentnon-labeled DNAs (including those corresponding to sequences identifiedin the PEG-3 promoter, such as TATA, PEA3, E2A, GRE, E2F, TRE, acutephase reactive regulating element, SP1, API₁, AP2 and NF1) can be usedas competitors in the gel shift assay to determine the relationshipbetween the trans-acting factors and previously identifiedtranscriptional regulators. It is possible that the trans-acting factorsregulating transcriptional control of the PEG-3 promoter may be novel.To identify these factors extracts will be purified from E11 andE11-NMT, CREF and transformed CREF and untreated and DNA damaged CREFcells by two cycles of heparin-Sepharose column chromatography, twocycles of DNA affinity chromatography and separation onSDS-polyacrylamide gels (58,59). Proteins displaying appropriateactivity using gel shift assays will be digested in situ with trypsin,the peptides separated by HPLC and the peptides sequenced (60). Peptidesequences will be used to synthesize degenerate primers and RT-PCR willbe used to identify putative genes encoding the trans-acting factor.These partial sequences will be used with cDNA library screeningapproaches and the RACE procedure, if necessary, to identify full-lengthcDNAs encoding the trans-acting factors (17,47,61,62). Once identified,the role of the trans-acting factors in eliciting cancer progressionwill be analyzed. (i) The functionality of positive and negativetrans-acting factors will be determined by transiently and stablyexpressing these genes in E11 and E11-NMT cells to determine effects onanchorage independence and tumorigenic potential in nude mice (stableexpression). Positive effects would be indicated if overexpressing apositive trans-acting factor facilitates the progression phenotype,whereas overexpressing a negative trans-acting factor inhibits theprogression phenotype. (ii) Antisense approaches will be used todetermine if blocking the expression of positive or negativetrans-acting factors can directly modify the progression state. A directeffect of a positive trans-acting factor in affecting progression wouldbe suggested if antisense inhibition of the positive factor partially orcompletely inhibits the progression phenotype in E11-NMT, i.e., growthin agar is reduced and tumor latency time is extended. Conversely, adirect effect of negative trans-acting factors in inhibiting progressionwould be suggested if antisense inhibition of the negative factorenhances the ability of E11 to grow in agar and reduces tumor latencytime. A potential problem with these types of studies would beencountered if the factors are involved in the regulation of many genes,e.g., Fos/Jun, and the antisense effects may, therefore, benon-specific. Although not within the scope of the present proposal,depending on the results obtained, cis-element knockouts could be usedto further define the role of these elements in regulating PEG-3expression.

[0522] For DNase-I footprinting assays, nuclear extracts from E11 andE11-NMT, CREF and transformed CREF and untreated CREF and DNA damaged(MMS and gamma irradiation) CREF cells will be prepared and DNase-Ifootprinting assays will be performed as described (44,63,64). Thepromoter necessary for PEG-3 expression, identified from the experimentsdescribed above, will be terminally labeled with ³²P and incubated withcrude nuclear extracts from the different cell types and experimentalconditions described above using established protocols (44,63,64). Thereaction mixture that has been digested with DNase-I enzyme will beterminated and the digested products will be analyzed on an 8%sequencing gel. The differential protection between nuclear extractsfrom progressed versus unprogressed, untransformed and oncogenicallytransformed and undamaged and DNA damaged cells will provide relevantinformation concerning the involvement of trans-acting factors inactivation and the location of specific sequences in the cis-regulatoryelements of the PEG-3 promoter mediating this activation. Ifdifferential protection is not detected using this approach, thesensitivity of the procedure can be improved by using different sizedDNA fragments from the PEG-3 promoter region or by using partiallypurified nuclear extracts (44,63,64).

[0523] The studies described above will result in the characterizationof the PEG-3 promoter region, the identification of cis-actingregulatory elements in the PEG-3 promoter and the identification oftrans-acting regulatory elements that activate (or repress) expressionof the PEG-3 gene as a function of cancer progression, oncogenictransformation and DNA damage. This information could prove valuable indesigning approaches for selectively inhibiting PEG-3 expression, andtherefore modifying cellular phenotypes related to cancer progressionand response to DNA damage.

[0524] The PEG-3 promoter as a sensitive biosensor monitoring system foridentifying compounds with the capacity to modulate cancer progressionand oncogenic transformation. As documented in this grant proposal,PEG-3 expression is elevated as a function of cancer progression,oncogenic transformation and DNA damage (17). Moreover, the PEG-3promoter displays increased activity in cells displaying these differentphenotypes (FIGS. 15, 16 & 17). These observations suggest that cellcultures stably expressing PEG-3 may prove effective as biosensormonitoring systems for identifying compounds and experimental conditionsthat can regulate important physiological processes, including cancerprogression, oncogenic transformation, DNA damage and angiogenesis. Thisapproach is called the Rapid Promoter Screening (RPS) assay system (FIG.18). This strategy uses stable cell lines containing a PEG-Luciferasetransgene for evaluating the activation or suppression of transcriptionfrom the PEG-3 promoter. Stable expression systems are preferable totransfection of the PEG-Luciferase gene into target cells since they arenot dependent on variable transfection efficiencies or affected bycellular heterogeneity. Moreover, cells containing a stablePEG-Luciferase transgene can be used to develop a high throughput RPSassay system with the capacity to evaluate large numbers of compounds ina simple manual or automated assay. Initial proof-of-principle for theRPS assay system has now been obtained. CREF and 4NMT clones have beenestablished that stably express a PEG-Luciferase transgene (FIG. 17).Treatment of CREF-PEG-Luc clones with the DNA damaging agent MMS (100μg/ml) results in a temporal induction of luciferase activity (FIG. 17).Similarly, MMS treatment of parental CREF cells transiently transfectedwith a PEG-Luciferase construct results in similar kinetics ofluciferase induction. Stable PEG-Luciferase expressing clones of 4NMTcells, tumorigenic CREF-Trans 6 cells transformed with human LNCaP DNAand expressing the PTI-1 oncogene (65), results in 4NMT-PEG-Luc clonesconstitutively expressing luciferase activity (unpublished data). Whentreated with an antisense phosphorothioate oligonucleotide that modifiesthe transformed state by suppressing PTI-1 expression, the level ofluciferase expression is inhibited (unpublished data). These resultssupport the suggestion that this approach will prove amenable fordeveloping an RPS biosensor monitoring assay system to detect agentsinducing DNA damage, enhancing cancer progression, inducing oncogenictransformation pathways and regulating angiogenesis (CREF-PEG-Luc) oragents inhibiting these processes (4NMT-PEG-Luc).

[0525] Initial studies will focus on the CREF-PEG-Luc and 4NMT-PEG-Lucassay systems. These cells will be used to test the utility of the RPSbiosensor monitoring system with well characterized reagents capable ofmodifying specific physiological pathways. The two cell types will betreated with various agents, including DNA damaging (a spectrum ofcompounds that elicit different DNA repair pathways, used alone and incombination with agents that directly modify DNA repair processes),oncogenic transformation pathway inducing (such as phorbol ester tumorpromoters, tyrosine kinase pathway modifiers and compounds affecting DNAmethylation) and angiogenesis inducing and inhibiting agents (such asTNF-α, BFGF, alpha interferon, beta interferon, thrombospondin andpleiotropin) and then monitored for luciferase activity. If thesestudies are successful, i.e., the assay systems can identify agents thathave an impact on specific biological pathways, they would provide thebasis for future expanded studies using this strategy. These studieswould include: (i) Screening small molecules, produced byrecombinatorial chemistry, to identify potentially important andclinically useful modulators of DNA damage and repair, cancerprogression, oncogenic transformation and angiogenesis; (ii) Evaluatingstable CREF-ras-PEG-Luc, CREF-src-PEG-Luc, S CREF-HPV-PEG-Luc andadditional PEG-Luc transformed cell lines. These luciferase expressingcells could be used to directly identify inhibitory moleculessuppressing specific oncogenic transformation pathways. (iii)CREF-PEG-Luc or CREF-PEG-β-Gal (beta galactosidase) containing cellscould be used to identify transforming cDNAs capable of initiatingcellular transformation and consequently inducing PEG transcription. Thehuman tumor cDNA containing CREF-PEG-Luc or CREF-PEG-β-Gal expressingclones could then be used to identify the putative transforming humantumor cDNAs. (iv) Transfection of normal cDNAs into transformed CREF-PEGor CREF-β-Gal expressing cells could be used to identify potentiallynovel cancer suppressor genes, by their ability to inhibit PEGtranscription. Appropriately modified cells could then be used to clonethe putative human tumor suppressor cDNA. (v) A PEG-β-Gal transgenecould be inserted into mouse ovum to create transgenic mice harboringthis gene. These animals could then be used as a sensitive in vivoindicator for evaluating DNA damage resulting from exposure tochemotherapeutic agents, evaluating gene regulation leading to cancerformation and identifying early stages in the conversion of a normalcell into a cancer cell.

[0526] PEG-3 Promoter-Luciferase Biosensor Monitoring Assay System: Thebasic protocol for this assay will involve incubating target cells forvarying times with compounds or growing cells under experimentalconditions that either induce (CREF-PEG-Luc) or inhibit (4NMT-PEG-Luc)luciferase activity and assaying for such activity. As part of theexperimental procedures, appropriate control reagents will be used.These will include MMS for the CREF-PEG-Luc cell culture system andPTI-1 antisense phosphorothioate oligonucleotide bridge primers for the4NMT-PEG-LUC cell culture system. Two formats can be used for assayingcompounds, one employing cells plated in 35 mm-tissue culture plates andthe other employing cells plated in 96-well microtiter plates. Theformer approach will be used to evaluate small numbers of compounds orexperimental conditions (maximum of 60 plates (20 agents tested intriplicate) in a single assay), and the latter approach can be adaptedfor screening large numbers of compounds in an automated fashion (96agents tested per assay block, using multiple assay blocks). Afterincubating cells for different times with test reagents, growth mediumwill be removed, the cells will be washed with serum-free growth mediumand the cells will be lysed using Reporter Lysis Buffer (Promega, Cat #E4531). Samples (placed in microcentrifuge tubes) or plates (96-wellmicrotiter format) can be stored at −70° C. (to be assayed within 24-hror stored for several weeks with samples remaining stable throughseveral freeze-thaw cycles). Samples or plates are centrifuged to removedebris and a 10 μl aliquot is removed for monitoring luciferase assayusing a luminometer.

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[0577] 50. Su Z-z, Shen R, Young C S H & Fisher P B. Genetic analysis ofcarcinogen enhancement of type 5 adenovirus transformation oc clonedFischer rat embryo fibroblast cells. Mol Carcinog, 8: 155-166, 1993.

[0578] 51. Gorospe M, Martindale J L, Sheikh M S, Formace A J Jr &Holbrook N J. Regulation of p₂₁ ^(CIP1/WAF1) expression by cellularstress: p53-dependent and p53-independent mechanisms. Mol CellDifferent, 4 (1): 47-65, 1996.

[0579] 52. Maniatis T, Goodbourn S & Fischer A. Regulation of inducibleand tissue-specific gene expression. Science, 236: 1237-1244, 1987.

[0580] 53. Ptashne M. How eukaryotic transcriptional activators work.Nature, 335: 683-689, 1988.

[0581] 54. Su Z-z, Yemul S, Stein C A & Fisher P B. c-fos is a positiveregulator of carcinogen enhancement of adenovirus transformation.Oncogene, 10: 2037-2049, 1995.

[0582] 55. Jiang H, Lin J, Young S-m, Goldstein N I, Waxman S, Davila V,Chellappan S P & Fisher P B. Cell cycle gene expression and E2Ftranscription factor complexes in human melanoma cells induced toterminally differentiate. Oncogene, 11: 1179-1189, 1995.

[0583] 56. Thanos D & Maniatis T. The high mobility group proteinHMGI(Y) is required for NF-κB dependent virus induction of the humanIFN-b gene. Cell, 71: 777-789, 1992.

[0584] 57. Weber J A & Gilmour D S. Genomic footprinting of the hsp70and histone H3 promoters in Drosophila embryos reveal novel protein-DNAinteractions. Nucleic Acids Res, 23: 3327-3334, 1995.

[0585] 58. Kamat J P, Basu K, Satyamoorthy L, Showe L & Howe C C. IPEBtranscription factor regulating intracisternal A particle during F9 celldifferentiation is expressed at sites of lymphoid development. Mol RepDev, 41: 8-15, 1995.

[0586] 59. Basu A, Dong B, Krainer A R & Howe C C. The intracisternalA-particle proximal enhancer-binding protein activates transcription andis identical to the RNA- and DNA-binding protein p54^(nrb)/NonO. MolCell Biol, 17: 677-686, 1997.

[0587] 60. Aebersold R H, Leavitt R A, Saavedra R A, Hood L E & Kent S BH. Internal amino acid sequence analysis of proteins separated by one-or two-dimensional gel electrophoresis after in situ protease digestionon nitrocellulose. Proc Natl Acad Sci USA, 84: 6970-6974, 1987.

[0588] 61. Lin J J, Jiang H & Fisher P B. Characterization of a novelmelanoma differentiation associated gene, mda-9, that is down-regulatedduring terminal cell differentiation. Mol Cell Different, 4 (4):317-333, 1996.

[0589] 62. Lin J J, Jiang H & Fisher P B. Melanoma differentiationassociated gene-9, mda-9, is a human gamma interferon responsive gene.Gene, in press, 1997.

[0590] 63. Shen R, Goswami S K, Mascareno E, Kumar A & Siddiqui MAQ.Tissue-specific transcription of the cardiac myosin light-chain 2 geneis regulated by an upstream repressor element. Mol Cell Biol, 11:1676-1685, 1991.

[0591] 64. Fisher A L, Ohsako S & Caudy M. The WRPW motif of thehairy-related basic helix-loop-helix repressor proteins acts as a4-amino-acid transcription repression and protein-protein interactiondomain. Mol Cell Biol, 16: 2670-2677, 1996.

[0592] 65. Su Z-z, Goldstein N I & Fisher P B. Antisense inhibition ofthe PTI-1 oncogene reverses cancer phenotypes. Proc Natl Acad Sci USA,95: 1764-1769, 1998.

[0593] Sixth Series of Experiments

[0594] PEG-3 Promoter/PTI-1 System

[0595] Rationale: Cancer is a progressive, multigenic disordercharacterized by changes in the transformed phenotype that culminates inmetastatic disease. Through the use of subtraction hybridization, anovel gene associated with transformation progression in virus- andoncogene-transformed rat embryo cells was cloned. The gene, designatedPEG-3, shares significant nucleotide and amino acid sequence homologywith the hamster growth arrest and DNA-damage inducible gene gadd34 anda homologous murine gene, MyD116, that is induced during induction ofterminal differentiation by IL-6 in murine myeloid leukemia cells. PEG-3expression is elevated in rodent cells displaying aprogressed-transformed phenotype and in rodent cells transformed byvarious oncogenes including Ha-ras, v-src, mutant type 5 adenovirus, andhuman papilloma virus type 18 (HPV). PEG-3 is transcriptionallyactivated in rodent cells, as is gadd34 and MyD116, after treatment withDNA damaging agents. However, only PEG-3 is active in rodent cellsdisplaying a transformed progressed phenotype. PEG-3 has been shown tobe upregulated in ras, src, HPV, and PTI-1 transformed CREF-Trans 6cells but not in CREF-Trans 6 itself. In addition, the gene is alsoexpressed in various human tumor lines but not in normal cell lines.

[0596] Recently, the PEG-3 promoter has been isolated and linked to aluciferase reporter gene. Transient transfection of thepromoter/reporter construct into cell lines of both tumor and normalorigin has demonstrated that the promoter is constitutively expressed inthe tumor cell lines but not in normal cells. We have stably transfectedthe PEG-3/luciferase construct into various transformed derivatives ofCREF-Trans 6 containing the oncogenes PTI-1, ras, and src, as well cellstransformed by HPV. These cell lines are used as targets for our smallmolecule combinatorial libraries.

[0597] The PEG-3 Promoter Assay. Cell Lines: Stable transfectantsexpressing a PEG-3 promoter/luciferase construct were prepared inCREF-Trans 6:4NMT (PTI-1 positive), T24 (ras positive); CREF-src, andCREF-HPV. Clones were isolated that constitutively expressed high levelsof luciferase. These clones are currently being used as targets inGenQuest's small molecule screening assays.

[0598] The following protocol is being used to screen small moleculelibraries:

[0599] a. Cells (e.g., 4NMT; PTI-1 positive) are plated in 96 wellopaque microtiter plates at a cell concentration of 1-2×10⁴ cells/wellin 100 ul of growth medium.

[0600] b. A stock solution of the combinatorial library is prepared inDMSO (final concentration=1 mM). Further dilutions are made in growthmedium to final concentrations of 20 uM, 2 uM, and 0.2 uM.

[0601] c. 100 ul/well of each sample is added to the cells. The finalconcentrations are 10, 1, and 0.1 uM.

[0602] d. Growth medium alone is added to the first two wells (Row A,wells 1 and 2) of each plate; a positive control is added to the nexttwo wells (Row A, wells 3 and 4). In the case of PTI-1, an antisenseoligonucleotide to the bridge region has been shown to down-regulateluciferase activity within 24 hours.

[0603] e. Plates are incubated overnight at 37C. Medium is removed andcells are washed with PBS. Cells are lysed using Reporter Lysis.Buffersupplied by the kit manufacturer (Luciferase Reporter Gene Assay kit;Boehringer Mannheim, cat. #1 669 893).

[0604] f. Plates are placed ˜70C overnight. Alternatively, the platescan be stored for several weeks (the manufacturer notes that luciferaseis stable in their Reporter Lysis Buffer) over several freeze thawcycles).

[0605] 9. Plates are thawed and 50 ul of the luciferase reaction mixtureis added. Plates are immediately read in a 96 well luminometer (TropixTR717 microplate luminometer).

[0606] h. A hit is defined as the decrease in luciferase activitycompared to control.

[0607] This assay can be completed in 48 hours:

[0608] i. Overnight incubation with library

[0609] ii. Luciferase assay: 4 hours

[0610] For viability assays.

[0611] a. Plates are set up as above except in transparent 96 wellmicrotiter plates.

[0612] b. After a 24 hour incubation, 10 ul of WST-1 (Cell ProliferationReagent; Boehringer Mannheim 1 544 807) is added to each well.

[0613] c. Plates are read at 30, 60, 120, and 180 minutes in an ELISAreader at 450 nM).

[0614] d. Viability is calculated as a percentage of the untreatedcells.

[0615] Deletion Analysis of the Rat PEG-3 Promoter

[0616] A series of deletion mutants have been produced by mutagenesis(Erase-a-Base System, Promega), linked to a luciferase reporter gene andtransfected into different cell types to define the regions of the PEG-3promoter that mediate differential expression in progressed versusunprogressed, oncogenically transformed versus nontransformed and DNAdamaged versus undamaged cells. The mutagenesis procedure was performedas described by Promega. The position of the various deletion mutants(determined by sequence analysis) relative to the promoter are shown inFIG. 24. The various deletion clones are indicated by numbers rangingfrom 2 to 11, with 1 being the complete PEG-3 promoter, and the clonedesignations are indicated in brackets. These include: 1 (46): intactrat PEG-3 promoter; 2 (22): missing 223 nt from the 5′ region of the ratPEG-3 promoter; 3 (33): missing 386 nt from the 5′ region of the ratPEG-3 promoter; 4 (42): missing 507 nt from the 5′ region of the ratPEG-3 promoter; 5 (14): missing 784 nt from the 5′ region of the ratPEG-3 promoter; 6 (8): missing 809 nt from the 5′ region of the ratPEG-3 promoter; 7 (65): missing 853 nt from the 5′ region of the ratPEG-3 promoter; 8 (74): missing 1190 nt from the 5′ region of the ratPEG-3 promoter; 9 (17): missing 1215 nt from the 5′ region of the ratPEG-3 promoter; 10 (114): missing 1511 nt from the 5′ region of the ratPEG-3 promoter; and 11 (20): missing 1737 nt from the 5′ region of therat PEG-3 promoter. The different deletion mutants are being used todefine the regions of the PEG-3 promoter that can enhance or suppressexpression of the PEG-3 gene. These constructs can now be used todetermine the regulatory control of the PEG-3 gene includingautoregulation, developmental regulation, tissue and cell type specificexpression and differential expression in progressed versusunprogressed, enhanced expression as a function of oncogenictransformation and induction of expression as a consequence of DNAdamage.

[0617] Expression of the intact PEG-3 promoter and various deletionmutants of the PEG-3 promoter in the different cell types is shown inFIGS. 25 to 31. The different cell types include: E11 (FIG. 12), E11-NMT(FIG. 26), a comparison of E11 versus E11-NMT (FIG. 14), E11-PKC (FIG.28), CREF-HPV (FIG. 29), CREF-ras (FIG. 30) and CREF-Trans 6:4 NMT (FIG.31).

[0618] Seventh Series of Experiments

[0619] CURE (Cancer Utilized Reporter Execution): A Strategy forSelectively Inhibiting the Growth and/or Killing of Cancer Cells

[0620] Use of CIRAs (Cancer Inhibitory Recombinant Adenoviruses) inCURE. Cancer is a progressive process with defined temporal stagesculminating in metastatic potential by evolving tumor cells. Althoughextensively scrutinized the molecular determinants of cancer progressionremain unclear. Well-characterized cell culture systems are valuableexperimental tools for defining the biochemical and molecular basis ofprogression. Two rodent model systems are providing insights into thegenes and processes regulating malignant progression of the transformedcell.

[0621] In adenovirus type 5 (Ad5) transformed rat embryo (RE) cells,progression can occur spontaneously by tumor formation in nude mice orby ectopic expression of oncogenes and signal transducinggrowth-regulating genes. In all contexts of progression, thedemethylating agent 5-azacytidine (AZA) can reverse this processresulting in an unprogressed phenotype in >95% of treated clones.Inhibition of progression also occurs in this system after formingsomatic cell hybrids between progressed and unprogressed cells. Using animmortal cloned rat embryo fibroblast (CREF) cell culture system,progression to metastasis and reversion of progression can be regulatedby appropriate genetic manipulation using the Ha-ras oncogene and theKrev-1 suppressor gene. These experimental findings support thehypothesis that progression may involve the selective inactivation ofgenes that suppress progression (progression suppressing genes) and/orthe induction of genes that promote progression (progression enhancinggenes). Identification and characterization of these genetic elementswould prove of immense value for defining this significant and definingcomponent of the cancer process and could provide useful targetmolecules for intervening in the neoplastic process.

[0622] To elucidate the molecular basis of progression we are using asubtraction hybridization approach. Subtraction hybridization betweenprogressed and unprogressed Ad5 transformed RE cells resulted in thecloning of progression elevated gene-3, PEG-3, that displays coordinateexpression with the progression and transformation phenotypes in AdS andoncogene transformed rat embryo cultures. PEG-3 is a novel gene sharingnucleotide (˜73 and ˜68%) and amino acid (˜59 and ˜72%) sequencehomology with the hamster growth arrest and DNA damage inducible genegadd34 and a homologous murine gene, MyD116, that is induced duringinduction of differentiation by IL6 in murine myeloid leukemia cells.Like gadd34 and MyD116, PEG-3 expression is induced by DNA damage.Induction following DNA damage results from increased RNA transcriptionand elevated steady-state levels of the PEG-3 gene. PEG-3 expression isalso elevated in temperature sensitive mutant adenovirus transformedSprgaue-Dawley rat embryo cells as a consequence of increased expessionof the transformed phenotype, i.e., elevated cancer progression.Additionally, PEG-3 expression increases in CREF cells as a consequenceof oncogenic transformation by diverse acting oncogenes, includingHa-ras, v-src, human papilloma virus type 18, v-raf, mutant type 5adenovirus (H5hr1) and prostate tumor inducing gene-1 (PTI-1). Theseexperimental results support the hypothesis that PEG-3 expression isregulated by cancer progression, oncogenic transformation and DNAdamage.

[0623] To define the mode of action of PEG-3 a 5′ DNA sequencecontaining the promoter region of this gene has been isolated andanalyzed. The PEG-3-Promoter (PEG-Prom) (˜2.1 kb) has been linked to aluciferase reporter gene (PEG-Prom-Luc) and evaluated for expression invarious cell types. Elevated levels of PEG-Prom-Luc activity areapparent in transformed rodent cells displaying a progressed transformedphenotype, DNA damaged rodent and human cells, oncogenically transformedrodent cells and histologically distinct human cancer cells (includingmetastatic melanoma, glioblastoma multiforme and carcinomas of thebreast, cervix, colon, lung, nasopharyngx and prostate). On the basis ofthe selective activity of the PEG-Prom for cancer cells, genetic vectorsare being constructed that display targeted expression of growtharresting and apoptosis-inducing genes or genes encoding an enzymepermitting activation of a toxic product in cancer cells. Additionally,genetic vectors can be constructed using the CURE protocol that targetthe expression of molecules on the surface of only cancer cellspermitting the directed therapy of cancer using immunological reagents(monoclonal antibodies, cytotoxic T-cells, TILs, etc.) or toxicchemicals. These novel vectors are the basis for the CURE (CancerUtilized Reporter Execution) protocol (FIG. 32). As one application ofCURE, recombinant adenoviruses are being constructed that permit theefficient delivery of CURE vectors into cells. These vectors aredesigned as CIRAs (Cancer Inhibitory Recombinant Adenoviruses) and theycontain the PEG-Prom driving expression of target genes, includingwild-type p53 (wt p53), melanoma differentiation associated gene-7(mda-7), adenovirus E1A and E1B (Ad E1A and E1B) or herpes simplex type1 thymidine kinase gene (HSV TK) (FIG. 32). When cancer cells areinfected with the CIRAs, the appropriate genes are activated resultingin a direct growth inhibition or apoptosis (wt p53 or mda-7), cell deathfollowing adenovirus replication (Ad E1A and E1B) or cell deathfollowing administration of gangcyclovir (HSV TK).

[0624] The carcinogenic process involves a series of sequential changesin the phenotype of a cell resulting in the acquisition of newproperties or a further elaboration of transformation-associated traitsby the evolving tumor cell (rev 1-4). Although extensively studied, theprecise genetic mechanisms underlying tumor cell progression during thedevelopment of most human cancers remain unknown. Possible factorscontributing to transformation progression, include: activation ofcellular genes that promote the cancer cell phenotype, i.e., oncogenes;activation of genes that regulate genomic stability, i.e., DNA repairgenes; activation of genes that mediate cancer aggressiveness andangiogenesis, i.e., progression elevated genes; loss or inactivation ofcellular genes that function as inhibitors of the cancer cell phenotype,i.e., tumor and progression suppressor genes; and/or combinations ofthese genetic changes in the same tumor cell (rev 1-6). A useful modelfor defining the genetic and biochemical changes mediating tumorprogression is the Ad5/early passage RE cell culture system (1,7-15).Transformation of secondary RE cells by Ad5 is often a sequentialprocess resulting in the acquisition of and further elaboration ofspecific phenotypes by the transformed cell (7-10). Progression in theAd5-transformation model is characterized by the development of enhancedanchorage-independence and tumorigenic capacity (as indicated by areduced latency time for tumor formation in nude mice) by progressedcells (1,10). The progression phenotype in Ad5-transformed rat embryocells can be induced by selection for growth in agar or tumor formationin nude mice (7-10), referred to as spontaneous-progression, bytransfection with oncogenes (11,14), such as Ha-ras, v-src, v-raf orE6/E7 region of human papilloma virus type-18 (HPV-18), referred to asoncogene-mediated progression, or by transfection with specific signaltransducing genes (15), such as protein kinase C (PKC), referred to asgrowth factor-related, gene-induced progression.

[0625] Progression, induced spontaneously or after gene transfer, is astable cellular trait that remains undiminished in Ad5-transformed REcells even after extensive passage (>100) in monolayer culture(1,10,14). However, a single-treatment with the demethylating agent AZAresults in a stable reversion in transformation progression in >95% ofcellular clones (1,10,11,14,15). The progression phenotype is alsosuppressed in somatic cell hybrids formed between normal or unprogressedtransformed cells and progressed cells (12-14). These findings suggestthat progression may result from the activation of specificprogression-promoting (progression elevated) genes or the selectiveinhibition of progression-suppressing genes, or possibly a combinationof both processes. To identify potential progression inducing genes withelevated expression in progressed versus unprogressed AdS-transformedcells we are using a subtraction approach (14,16,17). The subtractionhybridization approach resulted in cloning of PEG-3 displaying elevatedexpression in progressed cells (spontaneous, oncogene-induced and growthfactor-related, gene-induced) than in unprogressed cells (parentalAd5-transformed, AZA-suppressed, and suppressed somatic cell hybrids)(17). These findings document a direct correlation between expression ofPEG-3 and the progression phenotype in this rat embryo model system.

[0626] The nucleotide sequence of PEG-3 is ˜73 and ˜68% and the aminoacid sequence is ˜59 and 72% homologous to gadd34 (18) and MyD116(19,20), respectively (17). The sequence homologies between PEG-3 andgadd34/MyD116 are highest in the amino terminal region of their encodedproteins, i.e., ˜69 and ˜76% homology with gadd34 and MyD116respectively, in the first 279 aa (17). In contrast, the sequence of thecarboxyl terminus of PEG-3 significantly diverges from gadd34/MyD116,i.e., only ˜28 and ˜49% homology in the carboxyl 88 aa (17). Thespecific function of the gadd34/MyD116 gene is not known. Like hamstergadd34 and its murine homologue MyD116, PEG-3 expression is induced inCREF cells by MMS and gamma irradiation (17). The gadd34/MyD116 gene, aswell as the gadd45, MyD118 and gadd153 genes, encode acidic proteinswith very similar and unusual charge characteristics (21). PEG-3 alsoencodes a putative protein with acidic properties similar to the gaddand MyD genes. The carboxyl-terminal domain of the murine MyD116 proteinis homologous to the corresponding domain of the herpes simplex virus 1γ₁34.5 protein, that prevents the premature shutoff of total proteinsynthesis in infected human cells (22,23). Replacement of thecarboxyl-terminal domain of γ₁34.5 with the homologous region fromMyD116 results in a restoration of function to the herpes viral genome,i.e., prevention of early host shutoff of protein synthesis (23).Although further studies are necessary, preliminary results indicatethat expression of a carboxyl terminus region of MyD116 results innuclear localization (23). Similarly, both gadd153 and gadd45 geneproducts are nuclear proteins (21). When transiently expressed invarious human tumor cell lines, gadd34/MyD116 is growth inhibitory andthis gene can synergize with gadd45 or gadd153 in suppressing cellgrowth (21). In contrast, ectopic expression of PEG-3 in normal CREF(cloned rat embryo fibroblast) and HBL-100 (normal breast epithelial)cells and cancer (E11 and E11-NMT (Ad5-transformed rat embryo) and MCF-7and T47D (human breast carcinoma) cells does not significantly inhibitcell growth or colony formation (17). These results suggest thatgadd34/MyD116, gadd45, gadd153 and MyD118, represent a novel class ofmammalian genes encoding acidic proteins that are regulated during DNAdamage and stress and involved in controlling cell growth. In thiscontext, PEG-3 would appear to represent an enigma, since it is notgrowth suppressive and its expression is elevated in cells displaying anin vivo proliferative advantage and a progressed transformed andtumorigenic phenotype (17). PEG-3 may represent a unique member of thisacidic protein gene family that directly functions in regulatingprogression, perhaps by constitutively inducing signals that wouldnormally only be induced during genomic stress. Additionally, PEG-3 maymodify the expression of down-stream genes involved in mediating canceraggressiveness, i.e., tumor- and metastasis-mediating genes and genesinvolved in tumor angiogenesis. In these contexts, PEG-3 could functionto modify specific programs of gene expression and alter genomicstability, thereby facilitating tumor progression. This hypothesis isamenable to experimental confirmation.

[0627] The final stage in tumor progression is the acquisition bytransformed cells of the ability to invade local tissue, survive in thecirculation and recolonize in a new area of the body, i.e., metastasis(rev. 24,25). Transfection of a Ha-ras oncogene into CREF cells (26)results in morphological transformation, anchorage-independence andacquisition of tumorigenic and metastatic potential (27-29).Ha-ras-transformed CREF cells exhibit profound changes in thetranscription and steady-state levels of genes involved in suppressionand induction of oncogenesis (30,31). Simultaneous overexpression of theHa-ras suppressor gene Krev-1 in Ha-ras-transformed CREF cells resultsin morphological reversion, suppression of agar growth capacity and adelay in in vivo oncogenesis (30). Reversion of transformation inHa-ras+Krev-1 transformed CREF cells correlates with a return in thetranscriptional and steady-state mRNA profile to that of nontransformedCREF cells (30,31). Following long latency times, Ha-ras+Krev-1transformed CREF cells form both tumors and metastases in athymic nudemice (30). The patterns of gene expression changes observed duringprogression, progression suppression and escape from progressionsuppression supports the concept of transcriptional switching as a majorcomponent of Ha-ras-induced transformation (30,31).

[0628] Analysis of PEG-3 expression in CREF cells and variousoncogene-transformed and suppressor gene-reverted CREF cells indicates adirect relationship between PEG-3 expression and transformation andoncogenic progression (17). Northern blotting indicates that CREF cellsdo not express PEG-3, whereas PEG-3 expression occurs in CREF cellstransformed by several diverse-acting oncogenes, including Ha-ras,v-src, HPV 18 and mutant Ad5 (H5hr1) (17). Suppression of Ha-ras-inducedtransformation by Krev-1 results in suppression of PEG-3 expression.However, both tumor-derived and metastasis-derived Krev-1Ha-ras-transformed CREF cells express PEG-3. The highest relative levelsof PEG-3 mRNA are consistently found in the metastasis-derivedHa-ras+Krev-1 transformed CREF cells. These results indicate a directrelationship between PEG-3 expression and the transformed and oncogeniccapacity of CREF cells. In addition, PEG-3 expression directlycorrelates with human melanoma progression, with the highest levels ofexpression found in metastatic human melanoma and reduced levelsobserved in normal human melanocytes, radial growth phase (RGP) primarymelanomas and early vertical growth phase (VGP) primary melanomas.

[0629] An important question is the role of PEG-3 in cancer progression.PEG-3 could simply correlate with transformation progression oralternatively it could directly regulate this process. To distinguishbetween these possibilities, E11 cells (not expressing PEG-3) weregenetically engineered to express PEG-3 (17). When assayed for growth inagar or aggressiveness in vivo in nude mice, E11-PEG-3 cells display aprogression phenotype akin to that seen in E11-NMT cells (17,31).Moreover, antisense inhibition of PEG-3 in E11-NMT (normally expressingPEG-3) results in suppression of the progression phenotype in vitro andin vivo (31). Although the mechanism by which PEG-3 affects cancerprogression in vivo remains to be determined, a potential role forinduction of angiogenesis by PEG-3 is suggested (31). Tumors isolatedfrom nude mice infected with E11-NMT and E11-PEG-3 clones are highlyvascularized and they contain large numbers of blood vessels, whereasE11 and E11-NMT-PEG-3 AS tumors grow slower and they remain compactwithout extensive blood vessel involvement (31). Further studies arenecessary to determine the potentially important relationship betweenPEG-3 expression and angiogenesis.

[0630] Defining the mechanism underlying the differential expression ofPEG-3 as a function of cancer progression, oncogenic transformation andDNA damage. Nuclear run-on assays indicate that PEG-3 expressiondirectly correlates with an increase in the rate of RNA transcription(17). This association is supported by the isolation of a genomicfragment upstream of the 5′ untranslated region of the PEG-3 cDNA anddemonstration that this sequence linked to a luciferase reporter gene isactivated as a function of cancer progression, oncogenic transformationand DNA damage. Additionally, changes in the stability of PEG-3 mRNA mayalso contribute to differential expression of this gene as a function ofcancer progression, oncogene expression and DNA damage. To address thisissue mRNA stability (RNA degradation) assays will be performed asdescribed in detail previously (32). Our analysis will focus on theeffect of cancer progression (E11-NMT, R1 and R2 cells), oncogenictransformation (Ha-ras, V-src, H5hr1 and HPV-18 transformed CREF cells)and DNA damage (gamma irradiation and MMS-treatment of CREF cells).Appropriate controls, E11, untransformed CREF cells and CREF cells nottreated with DNA damaging agents, respectively, and experimental sampleswill be incubated without additions or in the presence of 5 μg/ml ofactinomycin D (in the dark), and 30, 60 and 120 min later, totalcellular RNA will be isolated and analyzed for gene expression usingNorthern hybridization. RNA blots will be quantitated by densitometricanalysis using a Molecular Dynamics densitometer (Sunnyvale, Calif.)(32). These straight forward experiments will indicate if the stabilityof PEG-3 is altered in cells as a direct consequence of spontaneousprogression, expression of defined oncogenes or as a consequence of DNAdamage.

[0631] Most eukaryotic genes are regulated at the level of initiation ofgene transcription. Detailed characterization of many differenteukaryotic transcriptional units has led to the general concept thatspecific interactions of short DNA sequences, usually located at the5′-flanking region of the corresponding genes (cis-acting elements),with certain cellular proteins (trans-acting elements) play a major rolein determining the rate of initiation of gene transcription. Toelucidate the mechanism underlying the transcriptional regulation of thePEG-3 gene the 5′-flanking region of this gene is being analyzed. Theseexperiments are important and they will determine regulatory control ofthe PEG-3 gene including autoregulation, developmental regulation,tissue and cell type specific expression and differential expression inprogressed versus unprogressed cells, enhanced expression as a functionof oncogenic transformation and induction of expression as a consequenceof DNA damage. Once the appropriate regions of the PEG-3 gene regulatingthe initiation of transcription has been confirmed, studies will beconducted to determine the relevant trans-acting regulatory factors thatbind to specific cis-acting regulatory elements and activate or repressexpression of the PEG-3 gene. The experiments outlined below aredesigned to: [1] define the 5′-flanking regions of the PEG-3 geneinvolved in mediating differential activity of PEG-3 in progressed,oncogenically transformed and DNA damaged cells; [2] identify cis-actingregulatory elements in the promoter region of the PEG-3 gene which areresponsible for the differential induction of PEG-3 expression; and [3]identify and characterize trans-acting regulatory elements that activate(or repress) expression of the PEG-3 gene.

[0632] Primary analysis of the functional regions of the PEG-3 promoter.Using a genomic walking strategy we have identified a 5′-flankingpromoter region of the PEG-3 gene that appears to encompass afunctionally complete PEG-3 promoter. To define importanttranscriptional regulatory regions of the PEG-3 promoter, a heterologousexpression system containing a luciferase gene without promoter orenhancer has been developed using the full-length promoter construct(33-35). Internal deletion mutations will be generated either by takingadvantage of internal restriction sites or by a nested exonuclease IIIbase deletion strategy. These constructs will be transfected into E11and E11-NMT, untransformed and transformed CREF (HShr1, Ha-ras, v-srcand HPV-18) and control CREF and gamma irradiation or MMS treated CREFcells. On the basis of transfection analyses of various deletion andpoint mutations it will be possible to define elements responsible forinduction of PEG-3 as a consequence of cancer progression, specifictransformation pathways or DNA damage response.

[0633] Transcription of PEG-3 in E11-NMT cells, as determined by nuclearrun-on assays, is >20-fold higher than in E11 cells, whereas transienttransfection of the PEG-3 promoter-luciferase gene into these two celltypes indicates only an ˜5-fold increase in activity in E11-NMT versusE11 cells (FIG. 15). This could indicate that the PEG-3 gene isrepressed in non-expressing cells (such as E11) through a cis-actingmechanism that is non-functional on transiently transfected promoters.Various luciferase constructs will be transfected into the differentcell types by the lipofectamine method or electroporation (Gene Pulser,Bio-Rad) as previously described (33,36). To correct for DNA uptake andcell number used for each transfection experiment, the luciferaseconstructs will be transfected with plasmids containing bacterialβ-galactosidase gene under the control of an Rous sarcoma virus (RSV)promoter (33-35). Studies will be conducted using multiple adult rattissue Northern blots (CLONTECH) containing poly A⁺ RNA and probing withPEG-3 (as well as gadd34 and MyD116) to define which rat tissue normallyexpress PEG-3. Previous studies document that genes expressing in morethan one tissue often require different sequences flanking the 5′-end ofthe gene. It is possible that PEG-3 expression in any normal tissue orunder different circumstances in rat cells, i.e., progression, oncogenictransformation or DNA damage, may be regulated by different5′-sequences. In that case, we will obtain variable luciferaseactivities for different luciferase constructs in the various celllines. Transcription motifs contributing to PEG-3 regulation in atissue, cell type or specific progression, transformation or DNA damagepathway will thus be identified.

[0634] Identifying cis-acting elements in the PEG-3 promoter responsiblefor expression during cancer progression, oncogenic transformation andDNA damage. On the basis of the deletion studies described above, thepotential location of cis-acting elements responsible for expression ofPEG-3 during cancer progression, oncogenic transformation and DNA damagewill be identified. The ˜2.1 kb PEG-3 promoter has been sequenced andpotential regulatory elements have been identified by comparison topreviously characterized transcriptional motifs. The PEG-3 promotercontains a number of potentially important transcriptional motifsincluding PEA3 (AGGAAA), E2A (GCAGGTG), GRE (TGTTCT), E2F (TTTTGGCCG),TRE (GGTCA), acute phase reactive regulating element (GTGGGA), SP1(GGGCGG), AP1 (TGACTCA), AP2 (TCCCCAACCC) and NF1 (TGGATTTGAGCCA). Theimportance of these sequences in regulating PEG-3 expression duringcancer progression, oncogenic transformation and DNA damage will bedetermined by introducing point mutations in a specific cis element intothe promoter region using previously described site-specific mutagenesistechniques (33,37-40) or with recently described PCR-based strategies,i.e., ExSite™ PCR-based site-directed mutagenesis kit and the Chameleon™double-stranded site-directed mutagenesis kit (Stratagene, CA). Themutated promoter constructs will be cloned into luciferase expressionvectors and tested for their effects on the promoter function bytransfection into different cell types and monitoring luciferaseactivity. Since the promoter region for the PEG-3 gene is located infront of the luciferase reporter gene in the various pPEG-Luciferaseconstructs, the change in luciferase activity for each construct willpermit a direct comparison of the activity of the mutant promoter tothat of the unmodified PEG-3 promoter.

[0635] After the regulatory regions of the PEG-3 promoter are confirmedexperiments will be conducted to address a number of important questionsrelative to cancer progression, oncogenic transformation and DNA damageinduction of PEG-3 expression. (1) Nuclear run-on and transienttransfection assays with pPEG-Luciferase constructs will be used todetermine the effect of changes in DNA methylation (AZA and phenylbutyrate treatment) on PEG-3 expression in E11-NMT cells, treatment withdifferent classes of DNA damaging and cancer modulating agents (such asTPA, retinoids, UV-C, gamma irradiation, methylating carcinogens,topoisomerase inhibitors, okadaic acid, etc.) on PEG-3 expression inCREF and CREF-PEG-Luc cl 1 cells (PEG-Luciferase stably transformed CREFclone) and exposure to cancer modulating agents (such as the Krev-1gene, dominant negative inhibitors of specific oncogenes, chemicals suchas CAPE, retinoids, sodium butyrate, interferon, TNF-α and additionalprogression modulating agents) on PEG-3 expression in oncogenicallytransformed CREF cells (1,8-10,18,21,28,29,41-43); (2) The level ofPEG-3 transcription in cells displaying different stages of cancerprogression and oncogenic transformation, including rodent model systemsof cancer progression (such as the Dunning rat prostate model,metastatic murine melanoma variants, etc.) and additional rodent cellstransformed by various oncogenes. These studies will indicate ifexpression of PEG-3 occurs in additional pathways of progression andtransformation. (3) Transfection of varying lengths of the 5′ flankingregion and internal deletion luciferase constructs into rodent cellsdisplaying different stages of progression, transformed by differentclasses of oncogenes and treated with various DNA damaging and cancerpromoting and inhibiting agents. These regulatory elements will besequenced and compared with previously characterized transcriptionalmotifs to identify potential positive and negative regulatory elements;(4) In addition to mutagenesis studies (to define functional motifsregulating transcriptional regulation of the PEG-3 promoter),cotransfection studies will be conducted with cDNAs containing putativepositive acting regulatory elements and a minimal PEG-3promoter-Luciferase construct into unprogressed and progressed rodentcells, untransformed CREF and oncogenically transformed CREF anduntreated and DNA damage treated CREF cells. These studies will indicateif the introduction of specific putative positive acting regulatoryelements can enhance PEG-3 expression in cells cotransfected with aminimal PEG-3 promoter region. The potential role of putative cis-actingnegative regulatory elements will be addressed by cotransfection with acomplete PEG-3 promoter region into the same target cells. These studieswill provide relevant information about the potential role of inhibitoryelements in regulating PEG-3 expression. (5) Experiments will also beperformed to evaluate the status of the endogenous PEG-3 gene duringcancer progression, oncogenic transformation and DNA damage. This willbe approached by using DNase hypersensitivity assays to look forstructural changes in this gene (33). Although not within the scope ofthe present studies, future studies could involve the identification ofa human PEG-3 cDNA, elucidation of the human PEG-3 promoter and analysisof the level of PEG-3 expression in human progression model systems.These studies would be quite informative in providing a potential linkbetween PEG-3 expression and cancer progression in human cells.

[0636] Identifying trans-acting nuclear proteins that mediatetranscriptional enhancing activity of the PEG-3 gene during cancerprogression, oncogenic transformation and DNA damage. The current viewon regulation of eukaryotic gene expression suggests that trans-actingproteins bind to specific sites within cis-elements of a promoter regionresulting in transcriptional activation (44,45). Experiments will beperformed to identify trans-acting factors (nuclear proteins) anddetermine where these factors interact with cis-regulatory elements. Toachieve this goal, two types of studies will be performed, one involvinggel retardation (gel shift) assays (15,33,46,47) and the secondinvolving DNase-I footprinting (methylation interference) assays(33,48,49).

[0637] Gel shift assays will be used to analyze the interactions betweencis-acting elements in the PEG-3 promoter and trans-acting factors inmediating transcriptional control (15,46,47). For this assay,³²P-labeled cis-elements will be incubated with nuclear extracts fromE11 and E11-NMT, CREF and transformed CREF (Ha-ras, v-src, H5hr1 andHPV-18) and untreated CREF and CREF treated with MMS (100 μg/ml for 8hr) or gamma irradiation (10 Gy for 4 hr) and reaction mixtures will beresolved on 5 or 8% polyacrylamide gels. After autoradiography, thepattern of retarded DNAs on the gel will provide information concerningthe interaction between trans-acting factors and specific regions of thecis-acting elements in the PEG-3 promoter. Non-labeled cis-actingelements (self-competition) will be added as a competitor to duplicatesamples to eliminate the possibility of non-specific binding and toconfirm that the interaction is really conferred by the trans-actingfactor. To begin to identify the transacting factors, differentnon-labeled DNAs (including those corresponding to sequences identifiedin the PEG-3 promoter, such as TATA, PEA3, E2A, GRE, E2F, TRE, acutephase reactive regulating element, SP1, AP1, AP2 and NF1) can be used ascompetitors in the gel shift assay to determine the relationship betweenthe trans-acting factors and previously identified transcriptionalregulators. It is possible that the trans-acting factors regulatingtranscriptional control of the PEG-3 promoter may be novel. To identifythese factors extracts will be purified from E11 and E11-NMT, CREF andtransformed CREF and untreated and DNA damaged CREF cells by two cyclesof heparin-sepharose column chromatography, two cycles of DNA affinitychromatography and separation on SDS-polyacrylamide gels (50,51).Proteins displaying appropriate activity using gel shift assays will bedigested in situ with trypsin, the peptides separated by HPLC and thepeptides sequenced (52). Peptide sequences will be used to synthesizedegenerate primers and RT-PCR will be used to identify putative genesencoding the trans-acting factor. These partial sequences will be usedwith cDNA library screening approaches and the RACE procedure, ifnecessary, to identify full-length cDNAs encoding the trans-actingfactors (17,36,51,52). Once identified, the role of the trans-actingfactors in eliciting cancer progression will be analyzed. (1) Thefunctionality of positive and negative trans-acting factors will bedetermined by transiently and stably expressing these genes in E11 andE11-NMT cells to determine effects on anchorage independence andtumorigenic potential in nude mice (stable expression). Positive effectswould be indicated if overexpressing a positive trans-acting factorfacilitates the progression phenotype, whereas overexpressing a negativetrans-acting factor inhibits the progression phenotype. (2) Antisenseapproaches will be used to determine if blocking the expression ofpositive or negative trans-acting factors can directly modify theprogression state. A direct effect of a positive trans-acting factor inaffecting progression would be suggested if antisense inhibition of thepositive factor partially or completely inhibits the progressionphenotype in E11-NMT, i.e., growth in agar is reduced and tumor latencytime is extended. Conversely, a direct effect of negative trans-actingfactors in inhibiting progression would be suggested if antisenseinhibition of the negative factor enhances the ability of E11 to grow inagar and reduces tumor latency time. A potential problem with thesetypes of studies would be encountered if the factors are involved in theregulation of many genes, e.g., Fos/Jun, and the antisense effects may,therefore, be non-specific. Although not within the scope of the presentproposal, depending on the results obtained, cis-element knockouts couldbe used to further define the role of these elements in regulating PEG-3expression.

[0638] For DNase-I footprinting assays, nuclear extracts from E11 andE11-NMT, CREF and transformed CREF and untreated CREF and DNA damaged(MMS and gamma irradiation) CREF cells will be prepared and DNase-Ifootprinting assays will be performed as described (33,53,54). Thepromoter necessary for PEG-3 expression, identified from the experimentsdescribed above, will be terminally labeled with ³²P and incubated withcrude nuclear extracts from the different cell types and experimentalconditions described above using established protocols (33,53,54). Thereaction mixture that has been digested with DNase-I enzyme will beterminated and the digested products will be analyzed on an 8%sequencing gel. The differential protection between nuclear extractsfrom progressed versus unprogressed, untransformed and oncogenicallytransformed and undamaged and DNA damaged cells will provide relevantinformation concerning the involvement of trans-acting factors inactivation and the location of specific sequences in the cis-regulatoryelements of the PEG-3 promoter mediating this activation. Ifdifferential protection is not detected using this approach, thesensitivity of the procedure can be improved by using different sizedDNA fragments from the PEG-3 promoter region or by using partiallypurified nuclear extracts (33,53,54).

[0639] The studies described above will result in the characterizationof the PEG-3 promoter region, the identification of cis-actingregulatory elements in the PEG-3 promoter and the identification oftrans-acting regulatory elements that activate (or repress) expressionof the PEG-3 gene as a function of cancer progression, oncogenictransformation and DNA damage. This information could prove valuable indesigning approaches for selectively inhibiting PEG-3 expression, andtherefore modifying cellular phenotypes related to cancer progressionand response to DNA damage.

[0640] Isolation and initial characterization of the PEG-3 promoter.

[0641] Targeted adenovirus gene delivery system for selectivelyinhibiting proliferation or inducing toxicity in PEG-3 expressing cancercells: CURE (Cancer Utilized Reporter Execution) and CIRAs (CancerInhibitory Recombinant Adenoviruses). Gene based therapies that exploitdifferences between cancer cells and normal cells represent potentiallysignificant technologies for improved cancer therapy. This approach hasbeen used to selectively target the replication of an E1B, 55-kDagene-attenuated adenovirus (ONYX-015), to cancer cells containing amutant p53 gene (55,56). Moreover, a minimal promoter/enhancer constructderived from the 5′ flanking region of the human prostate specificantigen (PSA) promoter has been used to drive the expression of the Ad5E1A gene in a replication competent Ad, thereby selectively inducingviral replication and toxicity in PSA-expressing prostate cancer cells(57). Similarly, viruses expressing the herpes simplex thymidine kinase(TK) gene have been used in combination with gancyclovir or acyclovir totarget toxicity in cancer cells expressing herpes simplex viralthymidine kinase (58-61). In this context, a virus containing a genepromoter displaying restrictive or selective expression of a linked gene(with the capacity to inhibit growth or induce toxicity either directlyor indirectly) would represent an extremely valuable therapeuticreagent.

[0642] As documented experimentally, the PEG-3 promoter displayselevated expression in progressed cancer cells, oncogenicallytransformed cancer cells and DNA damaged cells. The absolute level ofinduction of luciferase activity in progressed cancer cells andoncogenically transformed cells is ≧10-fold higher than in DNA damagedCREF cells. In this respect, it is anticipated that the activity of thePEG-3 promoter will be reduced and less effective in driving a linkedgene in a recombinant replication competent or incompetent Ad whenexpressed in DNA damaged versus progressed or oncogenically transformedcells. However, it is likely that treatment with DNA damaging agentswill even further augment the activity of the PEG-3 promoter in cancercells. Moreover, preliminary experiments using a large panel of humancancer cell lines, including metastatic melanoma, glioblastomamultiforme and carcinomas of the breast, cervix, colon, lung,nasopharyngx and prostate, indicate that the PEG-3 promoter is active,whereas no activity is apparent in their normal cellular counterparts(unpublished data). On the basis of these considerations, the PEG-3promoter would appear to be an ideal genetic tool for the constructionof “cancer inhibitory recombinant adenoviruses” (CIRAs). These CIRAscould be used as part of a protocol called “cancer utilized reporterexecution” (CURE) to selectively induce growth suppression, apoptosis ortoxicity uniquely in cancer cells.

[0643] We propose to construct evaluate CURE using CIRAs that permitexpression of a gene inhibiting growth and or inducing toxicityspecifically in cancer cells using the PEG-3 promoter to control geneexpression. Additionally, the PEG-3 promoter can be used to driveexpression of a gene encoding an antigenic epitope that will increasethe immunogenicity and killing of modified cells by the immune system(activated T-cells and/or antibodies). Several types of CIRAs will begenerated and tested for biological efficacy using the CURE protocol.

[0644] 1. Recombinant Ad expressing a wild-type p53 gene controlled bythe PEG-3 promoter, Ad.PEG-wtp53. These viruses, using conventionalcytomegalovirus promoters to drive wild-type p53 expression, are provingefficacious in treating a number of tumor types in humans (62). It ispredicted that Ad.PEG-wtp53 would inhibit the growth or induce apoptosisin cancer cells containing defects in p53, which occur in a very highpercentage of cancer cells (63). Moreover, even if low level PEG-3promoter activity occurs in normal cells, the subsequent low level ofp53 expression should not significantly affect cell growth or viabilityin normal cells.

[0645] 2. Recombinant Ad expressing the novel mda-7 cancer growthsuppressor gene controlled by the PEG-3 promoter, Ad.PEG-mda-7. Themda-7 gene is selectively growth suppressive in cancer versus normalcells (64). Moreover, the inhibitory effect of mda-7 in tumor cells isindependent of p53 status and occurs in cancer cells with diversegenetic defects (64). This novel tumor suppressor gene would appearideally suited for therapeutic applications (CURE) and the generation ofCIRAs, since expression in normal cells (even following infection with100 pfu/cell of Ad.mda-7 S virus) does not elicit a biological phenotype(65).

[0646] 3. Recombinant Ad expressing the cyclin-dependent kinaseinhibitor p21 (66) controlled by the PEG-3 promoter, Ad.PEG-p21. The p21gene can induce growth arrest and apoptosis in specific cancer cells(67). In addition, recent studies indicate that an Ad expressing p21 canbe used to inhibit tumor growth in animals (68). Since p21 can alsomodify growth in normal cells, the use of the PEG-3 promoter toselectively drive p21 in cancer cells would appear to be preferablevehicle for gene therapy than a virus constitutively expressing p21.

[0647] 4. Recombinant replication competent Ad expressing the viral E1Agene controlled by the PEG-3 promoter, Ad.PEG-E1A. In principle, thisvirus should only replicate in cancer cells that allow activation of thePEG-3 promoter. The successful application of this type of virus forcancer therapy will be contingent upon sufficient E1A expression topermit switch-on of additional Ad genes permitting virus replication andcytotoxicity only in the cancer cells. A potential problem that might beencountered using this type of CIRA is minimal activity of the PEG-3promoter resulting in low levels of Ad5 E1A expression in normal cells.This could prove problematic since previous studies have documented thateven very low levels of Ad E1A can result in virus production (69).Moreover, since CREF cells are nonpermissive for Ad replication (1,27),this type of CIRA requires testing in a human cell line. This could beaccomplished by using 293 cells, a human embryonic kidney cell linetransformed by sheared Ad5 and constitutively expressing the Ad5 E1A andE1B genes. Preliminary studies indicate that the PEG-3 promoter is veryactive in 293 cells, whereas it displays no activity in normal humanfibroblast or epithelial cells.

[0648] 5. Recombinant virus expressing a HSV 1 TK gene controlled by thePEG-3 promoter, Ad.PEG-TK. This virus should produce viral TK in cancercells as a result of activation of the PEG-3 promoter. By applyinggancyclovir or acyclovir it would be possible to selectively kill cancercells expressing elevated levels of viral TK. Previous studies provideprecedents for the use of the HSV 1 TK gene and gancyclovir or acyclovirto selectively kill cancer cells (58-61).

[0649] 6. Recombinant virus expressing an antigenic immunostimulatinggene, such as GM-CSF and/or IL-2, controlled by the PEG-3 promoter,Ad.PEG-ImStim. This virus can be used to infect cells resulting intargeted expression, because of PEG-3 promoter utilization, specificallyin cancer cells. The altered immunoreactivity in the tumor cells willelicit enhanced immune recognition and elimination of the tumor.

[0650] 7. Recombinant virus expressing a defined antigen (with andwithout a co-stimulatory molecule) controlled by the PEG-3 promoter,Ad.PEG-Antigen. This virus can be used to infect cells resulting intargeted expression, because of PEG-3 promoter utilization, of theantigen (with or without the co-stimulatory molecule) specifically incancer cells. The expression of this antigen on the surface of thecancer cells can then be used to target an antibody for immaging oftumor cells in a patient and/or inducing toxicity (by using an antibodywith cytotoxic properties, an antibody conjugated with a toxin or anantibody carrying a high energy emitting radionuclide). By usingappropriate vectors, all of the approaches briefly described above canalso be used to treat patients with systemic tumors and metastases.

[0651] The methodologies for construction of the different CIPAs areroutine and we have extensive experience in this approach (37-40,64). Wetherefore do not expect any problems in producing the appropriateviruses. Once the various recombinant viruses have been constructed theywill be evaluated for biological efficacy using in vitro assays (64) andif active using in vivo tumor xenograft models (27-29). As initial testsfor proof of practice of CURE and the CIRA approach, CREF, CREF-ras,CREF-src and CREF-HPV-18 cells will be infected with the specificrecombinant virus (Ad.PEG-wtp53, Ad.PEG-mda-7, Ad.PEG-p21 or Ad.PEG-TK)or a recombinant virus not containing any gene insert (Ad.vec) andcolony forming ability in monolayer culture will be determined (64). Asindicated above, to test the Ad.PEG-E1A construct we will use 293 andnormal human kidney cells. In the case of the Ad.PEG-TK virus system,infected cells will be cultured in the presence or absence ofgancyclovir or acyclovir. A biologically relevant endpoint would be astatistically significant reduction of colony formation in transformedcells, but not in normal cells, for the CIRA versus the Ad.vec. Thiscould be verified by monitoring expression of the transduced gene (RNAand protein levels) following viral infection. The levels of expressionof the transduced gene should be significantly higher in transformedcells versus parental CREF cells (or in 293 versus normal human kidneycells). If these results occur with any or all of the CIRAs, furtherstudies would be performed to assay for effects on tumor growth andmetastasis using previously described procedures (27-29). This wouldinclude injecting tumor cells infected with recombinant virus prior toinjection into animals and establishing tumors in animals followed byrepeated administration (2× per week) of recombinant virus. Ifsuccessful in reducing or eliminating cancer cells in vivo, the CIRAconcept could ultimately prove of immense value for the targeted therapyof human cancers. Obvious extensions of this approach would be toisolate a human PEG-3 promoter and construct recombinant viruses inwhich this promoter drives the gene of choice. It is realized that theuse of CIRAs for the therapy of human cancer depends on a number ofimportant considerations. The rat or human PEG-3 promoter must displaydifferential expression in human cancer versus normal human cells andthe level of expression of the PEG-3 promoter must be sufficiently highin the cancer cells to allow for expression of adequate amounts of thelinked gene to induce a biological effect in these cells. In preliminarystudies, the rat PEG-3 promoter displays differential expression inhuman cancer versus normal cells suggesting that it my provide theappropriate reagent for the CURE approach using CIRAs. Moreover, thelevel of expression in normal tissue must be negligible, although thisshould not be a problem when using Ad.PEG-wtp53, Ad.PEG-mda-7 orAd.PEG-p21.

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[0723] product causing the cell to express a specific antigen.

1 8 1 457 PRT Rat 1 Met Ala Pro Ser Pro Arg Pro Gln His Val Leu His TrpLys Glu Ala 1 5 10 15 His Ser Phe Tyr Leu Leu Ser Pro Leu Met Gly PheLeu Ser Arg Ala 20 25 30 Trp Ser Arg Leu Arg Gly Pro Glu Val Ser Glu AlaTrp Leu Ala Glu 35 40 45 Thr Val Ala Gly Ala Asn Gln Ile Glu Ala Asp AlaLeu Leu Thr Pro 50 55 60 Pro Pro Val Ser Glu Asn His Leu Pro Leu Arg GluThr Glu Gly Asn 65 70 75 80 Gly Thr Pro Glu Trp Ser Lys Ala Ala Gln ArgLeu Cys Leu Asp Val 85 90 95 Glu Ala Gln Ser Ser Pro Pro Lys Thr Trp GlyLeu Ser Asp Ile Asp 100 105 110 Glu His Asn Gly Lys Pro Gly Gln Asp GlyLeu Arg Glu Gln Glu Val 115 120 125 Glu His Thr Ala Gly Leu Pro Thr LeuGln Pro Leu His Leu Gln Gly 130 135 140 Ala Asp Lys Lys Val Gly Glu ValVal Ala Arg Glu Glu Gly Val Ser 145 150 155 160 Glu Leu Ala Tyr Pro ThrSer His Trp Glu Gly Gly Pro Ala Glu Asp 165 170 175 Glu Glu Asp Thr GluThr Val Lys Lys Ala His Gln Ala Ser Ala Ala 180 185 190 Ser Ile Ala ProGly Tyr Lys Pro Ser Thr Ser Val Tyr Cys Pro Gly 195 200 205 Glu Ala GluHis Arg Ala Thr Glu Glu Lys Gly Thr Asp Asn Lys Ala 210 215 220 Glu ProSer Gly Ser His Ser Arg Val Trp Glu Tyr His Thr Arg Glu 225 230 235 240Arg Pro Lys Gln Glu Gly Glu Thr Lys Pro Glu Gln His Arg Ala Gly 245 250255 Gln Ser His Pro Cys Gln Asn Ala Glu Ala Glu Glu Gly Gly Pro Glu 260265 270 Thr Ser Val Cys Ser Gly Ser Ala Phe Leu Lys Ala Trp Val Tyr Arg275 280 285 Pro Gly Glu Asp Thr Glu Glu Glu Glu Asp Ser Asp Leu Asp SerAla 290 295 300 Glu Glu Asp Thr Ala His Thr Cys Thr Thr Pro His Thr SerAla Phe 305 310 315 320 Leu Lys Ala Trp Val Tyr Arg Pro Gly Glu Asp ThrGlu Glu Glu Asp 325 330 335 Asp Gly Asp Trp Asp Ser Ala Glu Glu Asp AlaSer Gln Ser Cys Thr 340 345 350 Thr Pro His Thr Ser Ala Phe Leu Lys AlaTrp Val Tyr Arg Pro Gly 355 360 365 Glu Asp Thr Glu Glu Glu Asp Asp SerGlu Asn Val Ala Pro Val Asp 370 375 380 Ser Glu Thr Val Asp Ser Cys GlnSer Thr Gln His Cys Leu Pro Val 385 390 395 400 Glu Lys Thr Lys Gly CysGly Glu Ala Glu Pro Pro Pro Phe Gln Trp 405 410 415 Pro Ser Ile Tyr LeuAsp Arg Ser Gln His His Leu Gly Leu Pro Leu 420 425 430 Ser Cys Pro PheAsp Cys Arg Ser Gly Ser Asp Leu Ser Lys Pro Pro 435 440 445 Pro Gly IleArg Ala Leu Arg Phe Leu 450 455 2 590 PRT Rat 2 Met Ala Pro Ser Pro ArgPro Gln His Ile Leu Leu Trp Arg Asp Ala 1 5 10 15 His Ser Phe His LeuLeu Ser Pro Leu Met Gly Phe Leu Ser Arg Ala 20 25 30 Trp Ser Arg Leu ArgVal Pro Glu Ala Pro Glu Pro Trp Pro Ala Glu 35 40 45 Thr Val Thr Gly AlaAsp Gln Ile Glu Ala Asp Ala His Pro Ala Pro 50 55 60 Pro Leu Val Pro GluAsn His Pro Pro Gln Gly Glu Ala Glu Glu Ser 65 70 75 80 Gly Thr Pro GluGlu Gly Lys Ala Ala Gln Gly Pro Cys Leu Asp Val 85 90 95 Gln Ala Asn SerSer Pro Pro Glu Thr Leu Gly Leu Ser Asp Asp Asp 100 105 110 Lys Gln GlyGln Asp Gly Pro Arg Glu Gln Gly Arg Ala His Thr Ala 115 120 125 Gly LeuPro Ile Leu Leu Ser Pro Gly Leu Gln Ser Ala Asp Lys Ser 130 135 140 LeuGly Glu Val Val Ala Gly Glu Glu Gly Val Thr Glu Leu Ala Tyr 145 150 155160 Pro Thr Ser His Trp Glu Gly Cys Pro Ser Glu Glu Glu Glu Asp Gly 165170 175 Glu Thr Val Lys Lys Ala Phe Arg Ala Ser Ala Asp Ser Pro Gly His180 185 190 Lys Ser Ser Thr Ser Val Tyr Cys Pro Gly Glu Ala Glu His GlnAla 195 200 205 Thr Glu Glu Lys Gln Thr Glu Asn Lys Ala Asp Pro Pro SerSer Pro 210 215 220 Ser Gly Ser His Ser Arg Ala Trp Glu Tyr Cys Ser LysGln Glu Gly 225 230 235 240 Glu Ala Asp Pro Glu Pro His Arg Ala Gly LysTyr Gln Leu Cys Gln 245 250 255 Asn Ala Glu Ala Glu Glu Glu Glu Glu AlaLys Val Ser Ser Leu Ser 260 265 270 Val Ser Ser Gly Asn Ala Phe Leu LysAla Trp Val Tyr Arg Pro Gly 275 280 285 Glu Asp Thr Glu Asp Asp Asp AspSer Asp Trp Gly Ser Ala Glu Glu 290 295 300 Glu Gly Lys Ala Leu Ser SerPro Thr Ser Pro Glu His Asp Phe Leu 305 310 315 320 Lys Ala Trp Val TyrArg Pro Gly Glu Asp Thr Glu Asp Asp Asp Asp 325 330 335 Ser Asp Trp GlySer Ala Glu Glu Glu Gly Lys Ala Leu Ser Ser Pro 340 345 350 Thr Ser ProGlu His Asp Phe Leu Lys Ala Trp Val Tyr Arg Pro Gly 355 360 365 Glu AspThr Glu Asp Asp Gln Asp Ser Asp Trp Gly Ser Ala Glu Lys 370 375 380 AspGly Leu Ala Gln Thr Phe Ala Thr Pro His Thr Ser Ala Phe Leu 385 390 395400 Lys Thr Trp Val Cys Cys Pro Gly Glu Asp Thr Glu Asp Asp Asp Cys 405410 415 Glu Val Val Val Pro Glu Asp Ser Glu Ala Ala Asp Pro Asp Lys Ser420 425 430 Pro Ser His Glu Ala Gln Gly Cys Leu Pro Gly Glu Gln Thr GluGly 435 440 445 Leu Val Glu Ala Glu His Ser Leu Phe Gln Val Ala Phe TyrLeu Pro 450 455 460 Gly Glu Lys Pro Ala Pro Pro Trp Thr Ala Pro Lys LeuPro Leu Arg 465 470 475 480 Leu Gln Arg Arg Leu Thr Leu Leu Arg Thr ProThr Gln Asp Gln Asp 485 490 495 Pro Glu Thr Pro Leu Arg Ala Arg Lys ValHis Phe Ser Glu Asn Val 500 505 510 Thr Val His Phe Leu Ala Val Trp AlaGly Pro Ala Gln Ala Ala Arg 515 520 525 Arg Gly Pro Trp Glu Gln Leu AlaArg Asp Arg Ser Arg Phe Ala Arg 530 535 540 Arg Ile Ala Gln Ala Glu GluLys Leu Gly Pro Tyr Leu Thr Pro Ala 545 550 555 560 Phe Arg Ala Arg AlaTrp Ala Arg Leu Gly Asn Pro Ser Leu Pro Leu 565 570 575 Ala Leu Glu ProIle Cys Asp His Thr Phe Phe Pro Ser Gln 580 585 590 3 657 PRT Rat 3 MetAla Pro Ser Pro Arg Phe Gln His Val Leu His Trp Arg Asp Ala 1 5 10 15His Asn Phe Tyr Leu Leu Ser Pro Leu Met Gly Leu Leu Ser Arg Ala 20 25 30Trp Ser Arg Leu Arg Gly Pro Glu Val Pro Glu Ala Trp Leu Ala Lys 35 40 45Thr Val Thr Gly Ala Asp Gln Ile Glu Ala Ala Ala Leu Leu Thr Pro 50 55 60Thr Pro Val Ser Gly Asn Leu Leu Pro His Gly Glu Thr Glu Glu Ser 65 70 7580 Gly Ser Pro Glu Gln Ser Gln Ala Ala Gln Arg Leu Cys Leu Val Glu 85 9095 Ala Glu Ser Ser Pro Pro Glu Thr Trp Gly Leu Ser Asn Val Asp Glu 100105 110 Tyr Asn Ala Lys Pro Gly Gln Asp Asp Leu Arg Glu Lys Glu Met Glu115 120 125 Arg Thr Ala Gly Lys Ala Thr Leu Gln Pro Ala Gly Leu Gln GlyAla 130 135 140 Asp Lys Arg Leu Gly Glu Val Val Ala Arg Glu Glu Gly ValAla Glu 145 150 155 160 Pro Ala Tyr Pro Thr Ser Gln Leu Glu Gly Gly ProAla Glu Asn Glu 165 170 175 Glu Asp Gly Glu Thr Val Lys Thr Tyr Gln AlaSer Ala Ala Ser Ile 180 185 190 Ala Pro Gly Tyr Lys Pro Ser Thr Pro ValPro Phe Leu Gly Glu Ala 195 200 205 Glu His Gln Ala Thr Glu Glu Lys GlyThr Glu Asn Lys Ala Asp Pro 210 215 220 Ser Asn Ser Pro Ser Ser Gly SerHis Ser Arg Ala Trp Glu Tyr Tyr 225 230 235 240 Ser Arg Glu Lys Pro LysGln Glu Gly Glu Ala Lys Val Glu Ala His 245 250 255 Arg Ala Gly Gln GlyHis Pro Cys Arg Asn Ala Glu Ala Glu Glu Gly 260 265 270 Gly Pro Glu ThrThr Phe Val Cys Thr Gly Asn Ala Phe Leu Lys Ala 275 280 285 Trp Val TyrArg Pro Gly Glu Asp Thr Glu Glu Glu Asp Asn Ser Asp 290 295 300 Ser AspSer Ala Glu Glu Asp Thr Ala Gln Thr Gly Ala Thr Pro His 305 310 315 320Thr Ser Ala Phe Leu Lys Ala Trp Val Tyr Arg Pro Gly Glu Asp Thr 325 330335 Glu Glu Glu Asp Ser Asp Ser Asp Ser Ala Glu Glu Asp Thr Ala Gln 340345 350 Thr Gly Ala Thr Pro His Thr Ser Ala Phe Leu Lys Ala Trp Val Tyr355 360 365 Arg Pro Gly Glu Asp Thr Glu Glu Glu Asn Ser Asp Leu Asp SerAla 370 375 380 Glu Glu Asp Thr Ala Gln Thr Gly Ala Thr Pro His Thr SerAla Phe 385 390 395 400 Leu Lys Ala Trp Val Tyr Arg Pro Gly Glu Asp ThrGlu Glu Glu Asn 405 410 415 Ser Asp Leu Asp Ser Ala Glu Glu Asp Thr AlaGln Thr Gly Ala Thr 420 425 430 Pro His Thr Ser Pro Phe Leu Lys Ala TrpVal Tyr Arg Pro Gly Glu 435 440 445 Asp Thr Glu Asp Asp Thr Glu Glu GluGlu Asp Ser Glu Asn Val Ala 450 455 460 Pro Gly Asp Ser Glu Thr Ala AspSer Ser Gln Ser Pro Cys Leu Gln 465 470 475 480 Pro Gln Arg Cys Leu ProGly Glu Lys Thr Lys Gly Arg Gly Glu Glu 485 490 495 Pro Pro Leu Phe GlnVal Ala Phe Tyr Leu Pro Gly Glu Lys Pro Glu 500 505 510 Ser Pro Trp AlaAla Pro Lys Leu Pro Leu Arg Leu Gln Arg Arg Leu 515 520 525 Arg Leu PheLys Ala Pro Thr Arg Asp Gln Asp Pro Glu Ile Pro Leu 530 535 540 Lys AlaArg Lys Val His Phe Ala Glu Lys Val Thr Val His Phe Leu 545 550 555 560Ala Val Trp Ala Gly Pro Ala Gln Ala Ala Arg Arg Gly Pro Trp Glu 565 570575 Gln Phe Ala Arg Asp Arg Ser Arg Phe Ala Arg Arg Ile Ala Gln Ala 580585 590 Glu Glu Lys Leu Gly Pro Tyr Leu Thr Pro Asp Ser Arg Ala Arg Ala595 600 605 Trp Ala Arg Leu Arg Asn Pro Ser Leu Pro Gln Ser Glu Pro ArgSer 610 615 620 Ser Ser Glu Ala Thr Pro Leu Thr Gln Asp Val Thr Thr ProSer Pro 625 630 635 640 Leu Pro Ser Glu Thr Pro Ser Pro Ser Leu Tyr LeuGly Gly Arg Arg 645 650 655 Gly 4 2137 DNA Rat 4 ctgcagtact tgtacattgctaaataaaga gagggactcc aggaggagca gcctgggtct 60 aagaggtagg cagaaggaggttttaggggc ctgagcacaa gcttgaggag agaaaggtta 120 ttaaaaagcc agacgcttacaggtctcaga agggctagcc agaaactgtg gctggggtta 180 aggaaagggt ttaagagtgtgggcttttgg ttctgaggat gtagaacgtg aatgttgaga 240 gaagaaccaa gtggcggagttgggtgtgag caatgctatt aggaatttga ggcagggatt 300 cacgcgctgc tgtgactattttttaacaat gactcagtgc tgtgacctga tactgtttcc 360 agagcgactt ctaaacaaattccccctttc taggccagac acatggcccc aagcccaaga 420 ccccagcatg tcctgcactggaaggaagcc cactctttct acctcctgtc tccactgatg 480 ggcttcctca gccgggcctggagccgcctg agggggcccg aggtctcaga ggcctggttg 540 gcagaaacag tagcaggagcaaaccagata gaggctgatg ctctgttgac gcctcccccg 600 gtctctgaaa atcacctacctctccgagag actgaaggaa atggaactcc tgaatggagt 660 aaagcagccc agaggctctgccttgatgtg gaagcccaaa gttcccctcc taaaacttgg 720 ggactttcag agtattgatgaacataatgg gaagccagga caagatggcc ttagagagca 780 agaagtggag cacacagctggcctgcctac actacagccc cttcacctgc aaggggcaga 840 taagaaagtt ggggaggtggtggctagaga agagggtgtg tccgagctgg cttaccccac 900 atcacactgg gagggtggtccagctgagga tgaagaggat acagaaaccg tgaagaaggc 960 tcaccaggcc tctgctgcttccatagctcc aggatataaa cccagcactt ctgtgtattg 1020 cccaggggag gcagaacatcgagccacgga ggaaaaagga acagacaata aggctgaacc 1080 ctcaggctcc cactccagagtctgggagta ccacactaga gagaggccta agcaggaggg 1140 agaaactaag ccagagcaacacagggcagg gcagagtcac ccttgtcaga atgcagaggc 1200 tgaggaagga ggacctgagacttctgtctg ttctggcagt gccttcctga aggcctgggt 1260 gtatcgccca ggagaggacacagaggagga agaagacagt gatttggatt cagctgagga 1320 agacacagct catacctgtaccacccccca tacaagtgcc ttcctgaagg cctgggtcta 1380 tcgcccagga gaggacacagaagaggaaga tgacggtgat tgggattcag ctgaggaaga 1440 cgcgtctcag agctgtaccaccccccatac aagtgccttc ctgaggcctg ggtctatcgc 1500 ccaggagagg acacagaagaggaagacgac agtgagaatg tggccccagt tgactcagaa 1560 acagttgact cttgccagagtacccagcat tgtctaccag tagagaagac caagggatgt 1620 ggagaagcag agccccctcccttccagtgg ccttctattt acctggacag aagccagcac 1680 caccttgggc tgcccctaagctgccccttc gactgcagaa gcggctcaga tctttcaaag 1740 cccccgcccg gaatcagggccctgagattc ctctgaaggg tagaaaggtg cacttctctg 1800 agaaagttac agtccatttccttgctgtct gggcaggacc agcccaggct gctcgtcgag 1860 gcccctggga gcagtttgcacgagatcgaa gccgctttgc tcgacgcatt gccgtcctcg 1920 tctcttccac tgcctgagccttgctcttcc actgaggcca cacccctcag ccaagatgtg 1980 accactccct ctccccttcccagtgaaatc cctcctccca gcctggactt gggaggaagg 2040 cgggctaagc ctgagtagttttttgtgtat tctatgagtg ttagtctctt aatacgaata 2100 tgtaacgcct tttgcatttgtaaaaaaaaa aaaaaaa 2137 5 457 PRT Rat 5 Met Ala Pro Ser Pro Arg Pro GlnHis Val Leu His Trp Lys Glu Ala 1 5 10 15 His Ser Phe Tyr Leu Leu SerPro Leu Met Gly Phe Leu Ser Arg Ala 20 25 30 Trp Ser Arg Leu Arg Gly ProGlu Val Ser Glu Ala Trp Leu Ala Glu 35 40 45 Thr Val Ala Gly Ala Asn GlnIle Glu Ala Asp Ala Leu Leu Thr Pro 50 55 60 Pro Pro Val Ser Glu Asn HisLeu Pro Leu Arg Glu Thr Glu Gly Asn 65 70 75 80 Gly Thr Pro Glu Trp SerLys Ala Ala Gln Arg Leu Cys Leu Asp Val 85 90 95 Glu Ala Gln Ser Ser ProPro Lys Thr Trp Gly Leu Ser Asp Ile Asp 100 105 110 Glu His Asn Gly LysPro Gly Gln Asp Gly Leu Arg Glu Gln Glu Val 115 120 125 Glu His Thr AlaGly Leu Pro Thr Leu Gln Pro Leu His Leu Gln Gly 130 135 140 Ala Asp LysLys Val Gly Glu Val Val Ala Arg Glu Glu Gly Val Ser 145 150 155 160 GluLeu Ala Tyr Pro Thr Ser His Trp Glu Gly Gly Pro Ala Glu Asp 165 170 175Glu Glu Asp Thr Glu Thr Val Lys Lys Ala His Gln Ala Ser Ala Ala 180 185190 Ser Ile Ala Pro Gly Tyr Lys Pro Ser Thr Ser Val Tyr Cys Pro Gly 195200 205 Glu Ala Glu His Arg Ala Thr Glu Glu Lys Gly Thr Asp Asn Lys Ala210 215 220 Glu Pro Ser Gly Ser His Ser Arg Val Trp Glu Tyr His Thr ArgGlu 225 230 235 240 Arg Pro Lys Gln Glu Gly Glu Thr Lys Pro Glu Gln HisArg Ala Gly 245 250 255 Gln Ser His Pro Cys Gln Asn Ala Glu Ala Glu GluGly Gly Pro Glu 260 265 270 Thr Ser Val Cys Ser Gly Ser Ala Phe Leu LysAla Trp Val Tyr Arg 275 280 285 Pro Gly Glu Asp Thr Glu Glu Glu Glu AspSer Asp Leu Asp Ser Ala 290 295 300 Glu Glu Asp Thr Ala His Thr Cys ThrThr Pro His Thr Ser Ala Phe 305 310 315 320 Leu Lys Ala Trp Val Tyr ArgPro Gly Glu Asp Thr Glu Glu Glu Asp 325 330 335 Asp Gly Asp Trp Asp SerAla Glu Glu Asp Ala Ser Gln Ser Cys Thr 340 345 350 Thr Pro His Thr SerAla Phe Leu Lys Ala Trp Val Tyr Arg Pro Gly 355 360 365 Glu Asp Thr GluGlu Glu Asp Asp Ser Glu Asn Val Ala Pro Val Asp 370 375 380 Ser Glu ThrVal Asp Ser Cys Gln Ser Thr Gln His Cys Leu Pro Val 385 390 395 400 GluLys Thr Lys Gly Cys Gly Glu Ala Glu Pro Pro Pro Phe Gln Trp 405 410 415Pro Ser Ile Tyr Leu Asp Arg Ser Gln His His Leu Gly Leu Pro Leu 420 425430 Ser Cys Pro Phe Asp Cys Arg Ser Gly Ser Asp Leu Ser Lys Pro Pro 435440 445 Pro Gly Ile Arg Ala Leu Arg Phe Leu 450 455 6 2111 DNA Human CDS(294)...(2027) PEG3 6 tgagattgac tcagttcgca gcttgtggaa gattacatgcgagaaaaagc gcgactccgc 60 atccctttgc cgggacagcc cttgcgacag cccgtgagacatcacgtccc cgagccccac 120 ctttgccggg acagcctttg cgacagcccg tgagacatcacgtccccgag ccccacgcct 180 gagggcgaca tgaacgcgct ggccttgaga gcaatccggacccacgaccg cttttggcaa 240 accgaaccgg acctccagcc cccggggtga cgcgcagcccgccggccaga cac atg 296 Met 1 gcc cca agc cca aga ccc gag cat gtc ctg cactgg aag gaa gcc cac 344 Ala Pro Ser Pro Arg Pro Glu His Val Leu His TrpLys Glu Ala His 5 10 15 tct ttc tac ctc ctg tct cca ctg atg ggc ttc ctcagc cgg gcc tgg 392 Ser Phe Tyr Leu Leu Ser Pro Leu Met Gly Phe Leu SerArg Ala Trp 20 25 30 agc cgc ctg agg ggg ccc gag gtc tca gag gcc tgg ttggca gaa aca 440 Ser Arg Leu Arg Gly Pro Glu Val Ser Glu Ala Trp Leu AlaGlu Thr 35 40 45 gta gca gga gca aac cag ata cag gct gat gct ctg ttg acgcct ccc 488 Val Ala Gly Ala Asn Gln Ile Gln Ala Asp Ala Leu Leu Thr ProPro 50 55 60 65 ccg gtc tct gaa aat cac cta cct ctc cga gag act gaa ggaaat gga 536 Pro Val Ser Glu Asn His Leu Pro Leu Arg Glu Thr Glu Gly AsnGly 70 75 80 act cct gaa tgg agt aaa gca gcc cag agg ctc tgc ctt gat gtggaa 584 Thr Pro Glu Trp Ser Lys Ala Ala Gln Arg Leu Cys Leu Asp Val Glu85 90 95 gcc caa agt tcc cct cct aaa act tgg gga ctt tca gat att gat gaa632 Ala Gln Ser Ser Pro Pro Lys Thr Trp Gly Leu Ser Asp Ile Asp Glu 100105 110 cat aat ggg aag cca gga caa gat ggc ctt aga gag caa gaa gtg gag680 His Asn Gly Lys Pro Gly Gln Asp Gly Leu Arg Glu Gln Glu Val Glu 115120 125 cac aca gct ggc ctg cct aca cta cag ccc ctt cac ctg caa ggg gca728 His Thr Ala Gly Leu Pro Thr Leu Gln Pro Leu His Leu Gln Gly Ala 130135 140 145 gat aag aaa gtt ggg gag gtg gtg gct aga gaa gag ggt gtg tccgag 776 Asp Lys Lys Val Gly Glu Val Val Ala Arg Glu Glu Gly Val Ser Glu150 155 160 ctg gct tac ccc aca tca cac tgg gag ggt ggt cca gct gag gatgaa 824 Leu Ala Tyr Pro Thr Ser His Trp Glu Gly Gly Pro Ala Glu Asp Glu165 170 175 gag gat aca gaa acc gtg aag aag gct cac cag gcc tct gct gcttcc 872 Glu Asp Thr Glu Thr Val Lys Lys Ala His Gln Ala Ser Ala Ala Ser180 185 190 ata gct cca gga tat aaa ccc agc act tct gtg tat tgc cca ggggag 920 Ile Ala Pro Gly Tyr Lys Pro Ser Thr Ser Val Tyr Cys Pro Gly Glu195 200 205 gca gaa cat cga gcc acg gag gaa aaa gga aca gac aat aag gctgaa 968 Ala Glu His Arg Ala Thr Glu Glu Lys Gly Thr Asp Asn Lys Ala Glu210 215 220 225 ccc tca ggc tcc cac tcc aga ttc tgg gag tac cac act agagag agg 1016 Pro Ser Gly Ser His Ser Arg Phe Trp Glu Tyr His Thr Arg GluArg 230 235 240 cct aag cag gag gga gaa act aag cca gag caa cac agg gcaggg cag 1064 Pro Lys Gln Glu Gly Glu Thr Lys Pro Glu Gln His Arg Ala GlyGln 245 250 255 agt cac cct tgt cag aat gca gag tct gag gaa gga gga cctgag act 1112 Ser His Pro Cys Gln Asn Ala Glu Ser Glu Glu Gly Gly Pro GluThr 260 265 270 tct gtc tgt tct ggc agt gcc ttc ctg aag gcc tgg gtg tatcgc cca 1160 Ser Val Cys Ser Gly Ser Ala Phe Leu Lys Ala Trp Val Tyr ArgPro 275 280 285 gga gag gac aca gag gag gaa gaa gac cct gat ttg gat tcagct gag 1208 Gly Glu Asp Thr Glu Glu Glu Glu Asp Pro Asp Leu Asp Ser AlaGlu 290 295 300 305 gaa gac aca gct cat acc tgt acc acc ccc cat aca agtgcc ttc ctg 1256 Glu Asp Thr Ala His Thr Cys Thr Thr Pro His Thr Ser AlaPhe Leu 310 315 320 aag gcc tgg gtc tat cgc cca gga gag gac aca gaa gaggaa gat gac 1304 Lys Ala Trp Val Tyr Arg Pro Gly Glu Asp Thr Glu Glu GluAsp Asp 325 330 335 ggt gat tgg gat tca gct gag gaa gac gca gct cag agctgt acc acc 1352 Gly Asp Trp Asp Ser Ala Glu Glu Asp Ala Ala Gln Ser CysThr Thr 340 345 350 ccc cat aca agt gcc ttc ctg aag gcc tgg gtc tat cgccca gga gag 1400 Pro His Thr Ser Ala Phe Leu Lys Ala Trp Val Tyr Arg ProGly Glu 355 360 365 gac aca gaa gag gaa gac gac agt gag aat gtg gcc ccagtt gac tca 1448 Asp Thr Glu Glu Glu Asp Asp Ser Glu Asn Val Ala Pro ValAsp Ser 370 375 380 385 gaa aca gtt gac tct tgc cag agt acc cag cat tgtcta cca gta gag 1496 Glu Thr Val Asp Ser Cys Gln Ser Thr Gln His Cys LeuPro Val Glu 390 395 400 aag acc aag gga tgt gga gaa gca gag ccc cct cccttc cag gtg gcc 1544 Lys Thr Lys Gly Cys Gly Glu Ala Glu Pro Pro Pro PheGln Val Ala 405 410 415 ttc tat tta cct gga cag aag cca gca cca cct tgggca gcc cct aag 1592 Phe Tyr Leu Pro Gly Gln Lys Pro Ala Pro Pro Trp AlaAla Pro Lys 420 425 430 ctg ccc ctt cga ctg cag aag cgg ctc aga tct ttcaaa gcc ccc gcc 1640 Leu Pro Leu Arg Leu Gln Lys Arg Leu Arg Ser Phe LysAla Pro Ala 435 440 445 cgg aat cag ggc cct gag att cct ctg aag ggt agaaag gtg cac ttc 1688 Arg Asn Gln Gly Pro Glu Ile Pro Leu Lys Gly Arg LysVal His Phe 450 455 460 465 tct gag aaa gtt aca gtc cat ttc ctt gct gtctgg gca gga cca gcc 1736 Ser Glu Lys Val Thr Val His Phe Leu Ala Val TrpAla Gly Pro Ala 470 475 480 cag gct gct cgt cga ggc ccc tgg gag cag tttgca cga gat cga agc 1784 Gln Ala Ala Arg Arg Gly Pro Trp Glu Gln Phe AlaArg Asp Arg Ser 485 490 495 cgc ttt gct cga cgc att gcc cag gca gag gagcag ctg ggt cct tac 1832 Arg Phe Ala Arg Arg Ile Ala Gln Ala Glu Glu GlnLeu Gly Pro Tyr 500 505 510 ctt acc cct gct ttc agg gcc aga gca tgg acacgc ctt aga aac cta 1880 Leu Thr Pro Ala Phe Arg Ala Arg Ala Trp Thr ArgLeu Arg Asn Leu 515 520 525 ccc ctt cct ctg tcg tcc tcg tct ctt cca ctgcct gag cct tgc tct 1928 Pro Leu Pro Leu Ser Ser Ser Ser Leu Pro Leu ProGlu Pro Cys Ser 530 535 540 545 tcc act gag gcc aca ccc ctc agc caa gatgtg acc act ccc tct ccc 1976 Ser Thr Glu Ala Thr Pro Leu Ser Gln Asp ValThr Thr Pro Ser Pro 550 555 560 ctt ccc agt gaa atc cct cct ccc agc ctggac ttg gga gga agg cgg 2024 Leu Pro Ser Glu Ile Pro Pro Pro Ser Leu AspLeu Gly Gly Arg Arg 565 570 575 ggc taagcctgag tagttttttg ttatttatttattttaatac gaaataaagc 2077 Gly cttttgattt gtagtgaaaa aaaaaaaaaa aaaa2111 7 578 PRT Human 7 Met Ala Pro Ser Pro Arg Pro Glu His Val Leu HisTrp Lys Glu Ala 1 5 10 15 His Ser Phe Tyr Leu Leu Ser Pro Leu Met GlyPhe Leu Ser Arg Ala 20 25 30 Trp Ser Arg Leu Arg Gly Pro Glu Val Ser GluAla Trp Leu Ala Glu 35 40 45 Thr Val Ala Gly Ala Asn Gln Ile Gln Ala AspAla Leu Leu Thr Pro 50 55 60 Pro Pro Val Ser Glu Asn His Leu Pro Leu ArgGlu Thr Glu Gly Asn 65 70 75 80 Gly Thr Pro Glu Trp Ser Lys Ala Ala GlnArg Leu Cys Leu Asp Val 85 90 95 Glu Ala Gln Ser Ser Pro Pro Lys Thr TrpGly Leu Ser Asp Ile Asp 100 105 110 Glu His Asn Gly Lys Pro Gly Gln AspGly Leu Arg Glu Gln Glu Val 115 120 125 Glu His Thr Ala Gly Leu Pro ThrLeu Gln Pro Leu His Leu Gln Gly 130 135 140 Ala Asp Lys Lys Val Gly GluVal Val Ala Arg Glu Glu Gly Val Ser 145 150 155 160 Glu Leu Ala Tyr ProThr Ser His Trp Glu Gly Gly Pro Ala Glu Asp 165 170 175 Glu Glu Asp ThrGlu Thr Val Lys Lys Ala His Gln Ala Ser Ala Ala 180 185 190 Ser Ile AlaPro Gly Tyr Lys Pro Ser Thr Ser Val Tyr Cys Pro Gly 195 200 205 Glu AlaGlu His Arg Ala Thr Glu Glu Lys Gly Thr Asp Asn Lys Ala 210 215 220 GluPro Ser Gly Ser His Ser Arg Phe Trp Glu Tyr His Thr Arg Glu 225 230 235240 Arg Pro Lys Gln Glu Gly Glu Thr Lys Pro Glu Gln His Arg Ala Gly 245250 255 Gln Ser His Pro Cys Gln Asn Ala Glu Ser Glu Glu Gly Gly Pro Glu260 265 270 Thr Ser Val Cys Ser Gly Ser Ala Phe Leu Lys Ala Trp Val TyrArg 275 280 285 Pro Gly Glu Asp Thr Glu Glu Glu Glu Asp Pro Asp Leu AspSer Ala 290 295 300 Glu Glu Asp Thr Ala His Thr Cys Thr Thr Pro His ThrSer Ala Phe 305 310 315 320 Leu Lys Ala Trp Val Tyr Arg Pro Gly Glu AspThr Glu Glu Glu Asp 325 330 335 Asp Gly Asp Trp Asp Ser Ala Glu Glu AspAla Ala Gln Ser Cys Thr 340 345 350 Thr Pro His Thr Ser Ala Phe Leu LysAla Trp Val Tyr Arg Pro Gly 355 360 365 Glu Asp Thr Glu Glu Glu Asp AspSer Glu Asn Val Ala Pro Val Asp 370 375 380 Ser Glu Thr Val Asp Ser CysGln Ser Thr Gln His Cys Leu Pro Val 385 390 395 400 Glu Lys Thr Lys GlyCys Gly Glu Ala Glu Pro Pro Pro Phe Gln Val 405 410 415 Ala Phe Tyr LeuPro Gly Gln Lys Pro Ala Pro Pro Trp Ala Ala Pro 420 425 430 Lys Leu ProLeu Arg Leu Gln Lys Arg Leu Arg Ser Phe Lys Ala Pro 435 440 445 Ala ArgAsn Gln Gly Pro Glu Ile Pro Leu Lys Gly Arg Lys Val His 450 455 460 PheSer Glu Lys Val Thr Val His Phe Leu Ala Val Trp Ala Gly Pro 465 470 475480 Ala Gln Ala Ala Arg Arg Gly Pro Trp Glu Gln Phe Ala Arg Asp Arg 485490 495 Ser Arg Phe Ala Arg Arg Ile Ala Gln Ala Glu Glu Gln Leu Gly Pro500 505 510 Tyr Leu Thr Pro Ala Phe Arg Ala Arg Ala Trp Thr Arg Leu ArgAsn 515 520 525 Leu Pro Leu Pro Leu Ser Ser Ser Ser Leu Pro Leu Pro GluPro Cys 530 535 540 Ser Ser Thr Glu Ala Thr Pro Leu Ser Gln Asp Val ThrThr Pro Ser 545 550 555 560 Pro Leu Pro Ser Glu Ile Pro Pro Pro Ser LeuAsp Leu Gly Gly Arg 565 570 575 Arg Gly 8 2614 DNA Rat 8 acatgggcacgcgtggtcga cggcccgggc tggctgggca acacgggttc agcccaggtt 60 tcatagtaagttccagacac tcctggaaaa acaatacagg tccctgacaa aagaaaaaac 120 aaaacaaaggaaacagaaac atgcgttttt aaaaaagaag gaggagactc catgaaggca 180 ggccttgggtggggtcactg cttctctgta cacaggagga gaattgccaa gatcttccgg 240 acagtgtggactatactgta agaccctctc aatacagaca gactggacag gcatagtgac 300 acatgcctttaatgcctgca gtactcagga ggaggtggca ggtggaacgg ctgttctttg 360 aggttcaagaccagcgtgga ctacagagtg agttccagga caggcagggc tacacagaaa 420 aatcctgtctgaaaacaaaa caaaacccag acagacacac caaaaacagc caagggacca 480 gagagatgggtcagggccta atcacttgct actctttgca gaggacccaa atttagttcc 540 tataaccctccatgagaagc ttcacaattg tctctaactc aattccaccc gtgttccgac 600 ctccatatgcaccagacatg atatactcac acatacgcac aaacacacac acacacacac 660 acacacacacacacacacac acacacacac ggaaaacata taaaataaag atttaaaaaa 720 tctttttcttttggccgggg tgtgtgggag agcatctgag ccatctcacc agcccagggt 780 gcagctctttttcttttttt cggagctggg gaccgaaccc agagccttgt gcttgctagg 840 caagtgctctaccactgagc taaatcccca accccggagc acgtctttaa tcccagaatc 900 aggaggtagaggtaatgaga tcccagtgag cccaaggtca gccgagtcta caaagtgagt 960 tccaggacagccagaactaa tcttggaaaa acaaacaagg gctggtgagg tggttcagta 1020 gttaagaacactggctgctc ttccagaggt cctgagttca ttctcagtaa ccacatggtg 1080 gggatctgatgcctgttctg gcatgcagat atacatgcag atagtgcact cctacattta 1140 aaaaaaaaagacataaataa tattttaaaa cattgggcgt tttgtcttct aataaaactt 1200 cactgctatcttctaataaa aattcactgc tagccgcggg gtgtggtggc cccatacctt 1260 taatcccaacaacttgagag gcagaggcag gcggaccttt gagtttgaag ctagcctggt 1320 ctacagagtgagttcaagat agccacggat agtcagaaag tcctgtttcg aacctctccc 1380 caaccaaatcactcctgtaa tcccagcact ctggaggcag tagcaggtta gtccctgctt 1440 ctcagagagaggagagagag agagagagag agagagagga gacacacaca cacagagaca 1500 gagaggagagagaaagagaa agagaatggg acagcatgtg actgcctgat gaagttggcg 1560 tgcttgctcaaaagttctgc gagattgacg gctctctgga tttgagccaa ggacacgcct 1620 gggaagccacggtgacctca caaggcccgg aatctccgcg agaatttcag tgttgttttc 1680 ctctctccacctttctcagg gacttccgaa actccgcctc tccggtgacg tcagatagcg 1740 ctcgtcagactataaactcc cgggtgatcg tgttggcgca gattgactca gttcgcagct 1800 tgtggaagattacatgcgag accccgcgcg actccgcatc cctttgccgg gacagccttt 1860 gcgacagcccgtgagacatc acgtccccga gccccagcct gagggcgaca tgaacgcgct 1920 ggccttgagagcaatccgga cccacgatcg cttttggcaa accgaaccgg accgaaccgg 1980 acctccagcccccggggtga cgcgcagtcg ccggtgagtg ggggatgggg cggcctttgg 2040 gggagtgctggggaggactt tctttggcga tggaggctag gagagtgttg tgggatctag 2100 gggagactggggaggaaccc agatttgagg aaacggcact gaaagccgga tgctttattt 2160 ggtccgagagaggagagccc aggtctagtc tctacattga agggcagggg tcctgaacta 2220 gaactgcagtacttgtacat tgctaaataa agagagggac tccaggagga gcagcctggg 2280 tctaagaggtaggcaggaga aggttttagg ggcctgagca caagcttgag gagagaaagg 2340 ttattaaaaagccagacgtt acaggtctca gaagggctag ccagaaactg tggcttgggg 2400 ttaaggaaagggtttaagag tgtgggcttt tggttctgag gatgtaggaa cgtgaatgtt 2460 gagagaagaaccaagtggcg gagttgggtg tgagcaatgc tattaggaat ttgaggcagg 2520 gattcacgctgctgtgacta ttttttaaca atgactcagt gctgtgacct gatactgttt 2580 ccagagcgacttctaaacaa attcccccct ttct 2614

25. A method of treating cancer in a subject, comprising: a.administering the vector of claim 14 to the subject; and b.administering gancyclovir or acyclovir to the subject.
 26. A method oftreating cancer in a subject, comprising: a. administering the vector ofclaim 24 to the subject; and b. administering an antibody or a fragmentof an antibody to the antigen of claim 24 to the subject.
 27. The methodof claim 26, wherein the antibody is toxic or linked to a toxicsubstance.
 28. The method of claim 26, wherein the antibody is labeledand used for tumor imaging.
 29. The method of claim 27, wherein theantibody is radioactive.
 30. A pharmaceutical composition comprising thevector of claim 2 and a carrier.